JP5215180B2 - CD19 antibody and method of use thereof - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed on June 20, 2005 U.S. Provisional Patent Application No. 60 / 692,531; filed December 8, 2005 U.S. Provisional Patent Application No. 60 / 748,956; and 2006 Claims the benefit of US Provisional Patent Application No. 60 / 804,083, filed June 6, 2005; all of which are hereby incorporated by reference in their entirety.

background
CD19 is a 95 kDa membrane receptor that is expressed early in B cell differentiation and continues to be expressed until the B cell is induced for its final differentiation (Pezzutto et al., (1987) J Immunol. 138 : 2793; Tedder et al. (1994) Immunol. Today 15 : 437). The extracellular domain of CD19 contains two C2-type immunoglobulin (IG) -like domains that are separated by a small potential disulfide-linked domain. The cytoplasmic domain of CD19 has a unique structure, but is highly conserved among humans, mice, and guinea pigs (Fujimoto et al., (1998) Semin Immunol. 10 : 267). CD19 is part of a protein complex found on the cell surface of B lymphocytes. This protein complex includes CD19, CD21 (complement receptor, type 2), CD81 (TAPA-1), and CD225 (Leu-13) (Fujimoto, supra).

CD19 is an important regulator of transmembrane signals in B cells. Increased or decreased cell surface density of CD19 affects B cell development and function, leading to diseases such as autoimmune diseases or hypogammaglobulinemia (Fujimoto, supra). The CD19 complex enhances the response of B cells to antigens in vivo through the cross-linking of two distinct signaling complexes found on the B cell membrane. Two signaling complexes associated with IgM and CD19 on the membrane activate phospholipase C (PLC) by different mechanisms. Cross-linking of CD19 and B cell receptors reduces the number of IgM molecules required to activate PLC (Fujimoto, supra; Ghetie, supra). In addition, CD19 functions as an adapter protein specialized for amplification of Arc family kinases (Hasegawa et al., (2001) J Immunol 167 : 3190).

  CD19 binding has been shown to both enhance and inhibit B cell activation and proliferation, depending on the amount of crosslinking that has occurred (Tedder, supra). CD19 is expressed in more than 90% of B-cell lymphomas and was thought to affect lymphoma growth in vitro and in vivo (Ghetie, supra). The antibody produced against CD19 was a mouse antibody. A disadvantage of using mouse antibodies for the treatment of human subjects is the human anti-mouse (HAMA) response when administered to patients. Accordingly, there is a need for improved therapeutic antibodies against CD19 that are more effective in treating and / or preventing diseases mediated by CD19.

Overview The present invention provides isolated monoclonal antibodies, particularly human monoclonal antibodies, that bind to CD19 and exhibit many desirable properties. These properties include high affinity binding to human CD19. Also provided are methods of treating various CD19 mediated diseases using the antibodies and compositions of the invention.

In one aspect, the present invention provides:
(A) binds to human CD19 with a K _D of 1 × 10 ⁻⁷ M or less; and (b) binds to Raji B cell tumor cells and Daudi B cell tumor cells.
It relates to an isolated monoclonal antibody or an antigen-binding portion or fragment thereof.

  Preferably, the antibody is a human antibody, but in alternative embodiments the antibody can be a murine antibody, a chimeric antibody or a humanized antibody.

In one embodiment, the antibody, binds to human CD19 5 × 10 ^-8 M or or less a K _D, at 2 × 10 ^-8 M or less a K _D of binding to human CD19, 1 × 10 ^-8 M or less a K _D of at binds to human CD19, 5 × 10 ^-9 M or less a K _D of at binds to human CD19, 4 × 10 ^-9 M or less a K _D in binds to human CD19, which binds to human CD19 with 3 × 10 ^-9 M or less a K _D of at binds to human CD19, or 2 × 10 ^-9 M or less a K _D.

In another embodiment, the invention cross-competes with a reference antibody for binding to CD19, (a) binds human CD19 with a K _D of 1 × 10 ⁻⁷ M or less; and (b) Raji B cell tumor An isolated monoclonal antibody or antigen-binding portion thereof is provided that binds to cells and Daudi B cell tumor cells. In various embodiments, the reference antibody comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8;
Or the reference antibody comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9;
Or the reference antibody comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10;
Or the reference antibody comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11;
Or the reference antibody comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12;
Or the reference antibody comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 13;
Or the reference antibody comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14;
Alternatively, the reference antibody comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15.

In another aspect, the invention provides an isolated monoclonal antibody or antigen-binding portion thereof comprising a heavy chain variable region that is the product of or derived from a human V _H 5-51 gene that specifically binds CD19. Regarding fragments. The invention also provides an isolated monoclonal antibody or antigen-binding portion or fragment thereof comprising a heavy chain variable region that is the product of or derived from a human V _H 1-69 gene that specifically binds to CD19. The present invention yet further specifically binds to CD19, comprising a light chain variable regions from there or in a product of the human V _K L18 gene, provides an isolated monoclonal antibody, or antigen-binding portion or fragment thereof. The present invention yet further specifically binds to CD19, comprising a light chain variable regions from there or in a product of the human V _K A27 gene, provides an isolated monoclonal antibody, or antigen binding portion thereof. The present invention yet further comprises a light chain variable region or derived from a to a product of the human V _K L15 gene, specifically binds to CD19, an isolated monoclonal antibody or antigen-binding portion or fragment thereof provide.

In a preferred embodiment, the present invention comprises (a) the heavy chain variable region of a human V _H 5-51 or 1-69 gene; and (b) the light chain variable region of human V _K L18, A27 or V _K L15; An isolated monoclonal antibody or antigen-binding portion or fragment thereof that specifically binds to CD19 is provided.

In another aspect, the invention includes a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences; and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences; (a) heavy chain variable The region CDR3 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 30, 31, 32, 33, 34, 35 and 36 and conservative modifications thereof; (b) the light chain variable region CDR3 sequence Comprising an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57 and 58 and conservative modifications thereof; (c) 1 × 10 ⁻⁷ M or less bound to human CD19 with K _D; and and (d) binds to Raji B-cell tumor cells and Daudi B-cell tumor cells, provides an isolated monoclonal antibody or antigen binding portion thereof.

  Preferably, the heavy chain variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 23, 24, 25, 26, 27, 28 and 29 and conservative modifications thereof; and the light chain The variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 44, 45, 46, 47, 48, 49 and 50 and conservative modifications thereof. Preferably, the heavy chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22 and conservative modifications thereof; and the light chain The variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 37, 38, 39, 40, 41, 42 and 43 and conservative modifications thereof.

The preferred combination is:
(A) the heavy chain variable region CDR1 comprising SEQ ID NO: 16;
(B) heavy chain variable region CDR2 comprising SEQ ID NO: 23;
(C) heavy chain variable region CDR3 comprising SEQ ID NO: 30;
(D) a light chain variable region CDR1 comprising SEQ ID NO: 37;
(E) a light chain variable region CDR2 comprising SEQ ID NO: 44; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 51;
including.

Another preferred combination is:
(A) the heavy chain variable region CDR1 comprising SEQ ID NO: 16;
(B) heavy chain variable region CDR2 comprising SEQ ID NO: 23;
(C) heavy chain variable region CDR3 comprising SEQ ID NO: 30;
(D) a light chain variable region CDR1 comprising SEQ ID NO: 37;
(E) a light chain variable region CDR2 comprising SEQ ID NO: 44; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 52;
including.

Another preferred combination is:
(A) the heavy chain variable region CDR1 comprising SEQ ID NO: 17;
(B) heavy chain variable region CDR2 comprising SEQ ID NO: 24;
(C) heavy chain variable region CDR3 comprising SEQ ID NO: 31;
(D) a light chain variable region CDR1 comprising SEQ ID NO: 38;
(E) a light chain variable region CDR2 comprising SEQ ID NO: 45; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 53;
including.

Another preferred combination is:
(A) the heavy chain variable region CDR1 comprising SEQ ID NO: 18;
(B) heavy chain variable region CDR2 comprising SEQ ID NO: 25;
(C) heavy chain variable region CDR3 comprising SEQ ID NO: 32;
(D) a light chain variable region CDR1 comprising SEQ ID NO: 39;
(E) a light chain variable region CDR2 comprising SEQ ID NO: 46; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 54;
including.

Another preferred combination is:
(A) the heavy chain variable region CDR1 comprising SEQ ID NO: 19;
(B) heavy chain variable region CDR2 comprising SEQ ID NO: 26;
(C) heavy chain variable region CDR3 comprising SEQ ID NO: 33;
(D) a light chain variable region CDR1 comprising SEQ ID NO: 40;
(E) a light chain variable region CDR2 comprising SEQ ID NO: 47; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 55;
including.

Another preferred combination is:
(A) the heavy chain variable region CDR1 comprising SEQ ID NO: 20;
(B) heavy chain variable region CDR2 comprising SEQ ID NO: 27;
(C) heavy chain variable region CDR3 comprising SEQ ID NO: 34;
(D) a light chain variable region CDR1 comprising SEQ ID NO: 41;
(E) a light chain variable region CDR2 comprising SEQ ID NO: 48; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 56;
including.

Another preferred combination is:
(A) the heavy chain variable region CDR1 comprising SEQ ID NO: 21;
(B) heavy chain variable region CDR2 comprising SEQ ID NO: 28;
(C) heavy chain variable region CDR3 comprising SEQ ID NO: 35;
(D) a light chain variable region CDR1 comprising SEQ ID NO: 42;
(E) a light chain variable region CDR2 comprising SEQ ID NO: 49; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 57;
including.

Another preferred combination is:
(A) the heavy chain variable region CDR1 comprising SEQ ID NO: 22;
(B) heavy chain variable region CDR2 comprising SEQ ID NO: 29;
(C) heavy chain variable region CDR3 comprising SEQ ID NO: 36;
(D) a light chain variable region CDR1 comprising SEQ ID NO: 43;
(E) a light chain variable region CDR2 comprising SEQ ID NO: 50; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 58;
including.

Other preferred antibodies of the invention or antigen binding portions thereof are:
(A) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7; and (b) SEQ ID NOs: 8, 9, 10, A light chain variable region comprising an amino acid sequence selected from the group consisting of 11, 12, 13, 14 and 15;
The antibody specifically binds to CD19.

  Preferred combinations include: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

  Another preferred combination comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9.

  Another preferred combination comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10.

  Another preferred combination comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11.

  Another preferred combination comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.

  Another preferred combination comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 13.

  Another preferred combination comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14.

  Another preferred combination comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15.

  In another aspect of the invention, there is provided an antibody or antibody binding portion or fragment thereof that competes for binding to CD19 with any of the antibodies described above.

  An antibody of the invention can be, for example, a full-length antibody, eg, an IgG1 or IgG4 isotype. Alternatively, the antibody can be an antibody fragment, such as a Fab, Fab ′ or Fab′2 antibody, or a single chain antibody.

  The invention also provides an immunoconjugate comprising an antibody of the invention or an antigen-binding portion thereof or a fragment thereof conjugated to a therapeutic agent such as a cytotoxin or a radioisotope. The invention also provides bispecific molecules comprising an antibody or antigen-binding portion of the invention bound to a second functional portion that has a different binding specificity than the antibody or antigen-binding portion or fragment thereof.

  Also provided are compositions comprising an antibody of the invention, an antigen-binding portion or fragment thereof, an immune complex, or a bispecific molecule and a pharmaceutically acceptable carrier.

  Nucleic acid molecules encoding the antibodies of the invention or antigen binding portions or fragments thereof are likewise included in the invention along with expression vectors containing such nucleic acids and host cells containing such expression vectors.

  Other features and advantages of the invention will be apparent from the following detailed description and examples which should not be construed as limiting. The contents of all references, Genbank registrations, patents and published patent applications cited throughout this application are specifically incorporated herein by reference.

DETAILED DESCRIPTION The present disclosure relates to isolated monoclonal antibodies, particularly human monoclonal antibodies, that specifically bind to CD19 with high affinity. In certain embodiments, the antibodies of the invention comprise specific structural features such as CDR regions derived from specific heavy and light chain germline sequences and / or comprising specific amino acid sequences. The present invention relates to isolated antibodies, methods of making such antibodies, immune complexes, and bispecific molecules comprising such antibodies, as well as antibodies, immune complexes, or bispecifics of the present invention. A pharmaceutical composition comprising a therapeutic molecule is provided. The invention also relates to methods of using antibodies to detect CD19 and to treat diseases associated with expression of CD19, such as B cell malignancies that express CD19. Thus, the present invention provides for treating B cell malignancies, such as in the treatment of non-Hodgkin lymphoma, chronic lymphocytic leukemia, follicular lymphoma, diffuse large B cell lymphoma, and multiple myeloma. Methods of using the anti-CD19 antibodies of the invention are provided.

  In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

  The term “CD19” refers to variants, isoforms, and species homologues of, for example, human CD19. Accordingly, human antibodies of the present disclosure may cross react with CD19 from species other than humans in certain cases. In certain embodiments, the antibody may be completely specific for one or more human CD19 proteins and may not exhibit species or other types of non-human cross-reactivity. The complete amino acid sequence of an exemplary human CD19 has Genbank accession number NM_001770 (SEQ ID NO: 79).

  The term “immune response” refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the cells or liver described above. In the case of pathogens invaded by it, cells or tissues infected with pathogens, cancerous cells, or autoimmune or pathological inflammation, selective damage or destruction of normal human cells or tissues, or from the human body Refers to the effect of disappearance.

  “Signal transduction pathway” refers to the biochemical association between various signaling molecules that play a role in the transmission of signals from one part of the cell to another part of the cell. As used herein, the phrase “cell surface receptor” includes, for example, molecules and molecules of molecules capable of receiving signals and transducing such signals across the cytoplasmic membrane of a cell. A complex is included. An example of a “cell surface receptor” of the present invention is the CD19 receptor.

The term “antibody” as referred to herein includes whole antibodies and any antigen-binding fragment (ie, “antigen-binding portion”), or single chains thereof. “Antibody” refers to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) joined together by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain includes a heavy chain variable region (abbreviated herein as V _H ) and a heavy chain constant region. The heavy chain constant region includes three domains C _H 1, C _H 2, and C _H 3. Each light chain includes a light chain variable region (abbreviated herein as _VL ) and a light chain constant region. The light chain constant region is comprised of one domain, C _L. The _VH and _VL regions are further subdivided with respect to highly variable regions called complementarity determining regions (CDRs) interspersed with regions called more conserved framework regions (FR). Each V _H and V _L includes three CDRs and four FRs. These are arranged in the following order from the amino terminus to the carboxy terminus. FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. Antibody constant regions may mediate immunoglobulin binding to host tissues or factors, including cells of various immune systems (eg, effector cells) and the first component of the classical complement system (C1q) .

As used herein, an “antigen-binding portion” (or simply “antibody portion”) of an antibody is one or more of the antibodies that retain the ability to specifically bind to an antigen (eg, CD19). Refers to a fragment. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the term “antigen-binding portion” of an antibody include (i) a monovalent fragment consisting of a Fab fragment, V _L , V _H , C _L , and C _H1 domains; (ii) F ( ab ') ₂ fragment, a bivalent fragment comprising two Fab fragments joined by a disulfide bridge at the hinge region; (iii) an Fab' fragment that is essentially an Fab with part of the hinge region (FUNDAMENTAL IMMUNOLOGY, Paul ed ., 3.sup.rd ed. 1993); (iv) Fd fragment consisting of V _H and C _H 1 domains; (v) Fv fragment consisting of V _L and V _H domains of one arm of the antibody, (Vi) a dAb fragment consisting of a V _H domain (Ward et al. (1989) Nature 341: 544-546); (vii) an isolated complementarity determining region (CDR); and (viii) a nanobody, a single variable A heavy chain variable region comprising a domain and two constant regions is included. In addition, the two domains of the Fv fragment, V _L and V _H, are encoded by different genes, but they are monovalent molecules in which the V _L and V _H regions are paired using recombinant methods. Can be joined by a synthetic linker that allows them to be made as a single protein chain (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423- 426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

  As used herein, an “isolated antibody” is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (eg, a single antibody that specifically binds to CD19). Isolated antibodies are substantially free of antibodies that specifically bind to antigens other than CD19). However, isolated antibodies that specifically bind to CD19 are cross-reactive with other antigens such as CD19 molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

  The term “monoclonal antibody” or “monoclonal antibody composition” as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

  As used herein, the term “human antibody” is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is similarly derived from a human germline immunoglobulin sequence. The human antibodies of the invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, mutations that have been randomized or site-directed mutagenesis in vitro, or somatically mutated in vivo). Good. However, the term “human antibody” described herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto a human framework sequence. Is intended.

  The term “human monoclonal antibody” refers to an antibody that exhibits a single binding specificity with variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, a human monoclonal antibody is obtained by fusing B cells obtained from a transgenic non-human animal, such as a transgenic mouse having a genome comprising a human heavy chain and light chain transgene, to an immortalized cell. Produced by the hybridoma containing.

As used herein, the term “recombinant human antibody” includes (a) an animal (eg, a mouse) that is transgenic or transchromosomal for a human immunoglobulin gene, or Antibodies isolated from hybridomas prepared therefrom (further described below), (b) isolated from host cells transformed to express human antibodies, eg, transfectoma Prepared, expressed, generated by any other means including antibodies, (c) antibodies isolated from recombinant complex human antibody libraries, and (d) splicing human immunoglobulin gene sequences to other DNA sequences All human anti-antigens prepared, expressed, produced or isolated by recombinant means, such as isolated antibodies It is included. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when using animals that are transgenic for human Ig sequences), and Thus, the amino acid sequences of the _VH and _VL regions of the recombinant antibody are related and derived from the human germline _VH and _VL sequences, but naturally occur within the human antibody germline range in vivo. It is an array that may not.

  As used herein, “isotype” refers to the antibody class (eg, IgM or IgG1) that is encoded by heavy chain constant region genes.

  The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody that binds specifically to an antigen”.

  The term “human antibody derivative” refers to any modified form of a human antibody, eg, a conjugate of an antibody and another substance or antibody.

  The term “humanized antibody” is intended to refer to an antibody in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been grafted onto a human framework sequence. Additional framework region modifications may be made within the human framework sequences.

  The term “chimeric antibody” refers to a variable region sequence derived from one species, such as an antibody in which the variable region sequence is derived from a murine antibody and the constant region sequence is derived from a human antibody, and the constant region sequence is It is intended to refer to an antibody derived from a species.

As used herein, an antibody that “binds specifically to human CD19” has a K _{D of} 1 × 10 ⁻⁷ M or less, more preferably a K _{D of} 5 × 10 ⁻⁸ M, against human CD19. Or less, more preferably K _D 3 × 10 ⁻⁸ M or less, more preferably K _D 1 × 10 ⁻⁸ M or less, even more preferably K _D 5 × 10 ⁻⁹ M or less It is intended to refer to an antibody that binds.

As used herein, the term “K _assoc ” or “K _a ” refers to the rate of association of a particular antibody-antigen interaction and is used herein as “K _dis ” or “K _d ”. Is intended to refer to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term “K _D ” is intended to refer to the dissociation constant obtained from the ratio of K _{d to} K _a (ie, K _d / K _a ), and as molarity (M) It is written. K _D values for antibodies can be determined using methods well established in the art. A preferred method for determining the K _D of an antibody is preferably by using surface plasmon resonance using a biosensor system such as a Biacore (R) system.

As used herein, the term "high affinity" for an IgG antibody is, K _D 1 × 10 ^-7 M or less for a target antigen, and more preferably 5 × 10 ^-8 M or less , Even more preferably refers to an antibody having 1 × 10 ⁻⁹ M or less, even more preferably 10 ⁻⁹ M or less. However, “high affinity” binding can vary for other antibody isotypes. For example, "high affinity" binding relates IgM isotype, K _D is 10 ^-6 M or less, more preferably 10 ^-7 M or less, even more preferably an antibody with 10 ^-8 M or less Point to.

  As used herein, the term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, eg mammals and non-mammals such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles and the like.

Anti-CD19 Antibodies The antibodies of the present invention are characterized by certain functional features or characteristics of the antibody. For example, the antibody specifically binds to human CD19. Preferably, the antibody of the present invention, with high affinity to CD19, binding, for example, K _D 1 × 10 ^-7 M or less. The anti-CD19 antibody of the present invention preferably exhibits one or more of the following characteristics.
_{(A) K D 1 × 10} -7 M or less in binds human CD19;
(B) Binds to Raji B cell tumor cells and Daudi B cell tumor cells.

Preferably, the antibody binds with K _D 5 × 10 ^-8 M or less for human CD19 binds with K _D 1 × 10 ^-8 M or less for human CD19, to human CD19 binds with K _D 5 × 10 ^-9 M or less Te, binds with K _D 4 × 10 ^-9 M or less for human ^{_{CD19, K D 3 × 10 -9}} M or to human CD19 It binds with less, or binds with a K _D 2 × 10 ^-9 M or less for human CD19, or binds with K _D 1 × 10 ^-9 M or less for human CD19.

  Standard assays for assessing antibody binding ability to CD19 are known in the art, including, for example, ELISA, Western blot, RIA and flow cytometry analysis. Suitable assay methods are described in detail in the examples. Antibody binding kinetics (eg, binding affinity) can also be assessed by standard assays known in the art, such as Scatchard or Biacore® system analysis. Raji (ATCC deposit no. CCL-86) cells or Daudi (ATCC deposit no. CCL-213) cells are publicly available providers to assess the binding of Raji B cell tumor cells or Daudi B cell tumor cells For example, from the American Type Culture Collection and can be used in standard assays such as flow cytometry analysis.

Monoclonal antibodies 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8
Preferred antibodies of the invention are human monoclonal antibodies 21D4, 21D4a, 47G4, 27F3, 3C10, isolated and structurally characterized as described in Examples 16, 17, 18, 19, 20, 21, and 22. 5G7, 13F1 and 46E8. 21D4,21D4a, the V _H amino acid sequences of 47G4,27F3,3C10,5G7,13F1 and 46E8, respectively, SEQ ID NO: shown in 1,1,2,3,4,5,6 and 7. 21D4,21D4a, the V _L amino acid sequences of 47G4,27F3,3C10,5G7,13F1 and 46E8, respectively, SEQ ID NO: shown in 8,9,10,11,12,13,14 and 15.

Assuming that each of these antibodies binds to CD19, the V _H and _VL sequences can be “mixed and matched” to create other anti-CD19 binding molecules of the invention. The CD19 binding of such “mixed and matched” antibodies can be tested using the binding assays described above and in the Examples (eg, ELISA). Preferably, when the V _H and V _L chains are mixed and matched, the V _H sequence of a particular V _H / V _L pair is exchanged for a structurally similar V _H sequence. Similarly, preferably the V _L sequence from a particular V _H / V _L pairing is exchanged for a structurally similar V _L sequence.

Accordingly, in one aspect, the invention provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to CD19, preferably human CD19, comprising:
(A) SEQ ID NO: Heavy chain variable region comprising an amino acid sequence selected from the group consisting of 1, 2, 3, 4, 5, 6, and 7;
(B) A light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15.
Preferred heavy and light chain combinations include:
(A) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; or (a) the amino acid sequence of SEQ ID NO: 1 And (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and (b) a SEQ ID A light chain variable region comprising the amino acid sequence of NO: 10; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11. Or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12; or (a) the amino acid of SEQ ID NO: 5 A heavy chain variable region comprising the sequence; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 13; or (a) comprising the amino acid sequence of SEQ ID NO: 6 A heavy chain variable region; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7; and (b) SEQ ID NO: : Light chain variable region comprising 15 amino acid sequences,
Is included.

In another aspect, the present invention provides antibodies or combinations thereof comprising 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8 heavy and light chain CDR1, CDR2 and CDR3. The amino acid sequences of the V _H CDR1s of 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8 are shown in SEQ ID NOs: 16, 17, 18, 19, 20, 21 and 22. The amino acid sequences of the V _H CDR2s of 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8 are shown in SEQ ID NOs: 23, 24, 25, 26, 27, 28 and 29. The amino acid sequences of the V _H CDR3s of 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8 are shown in SEQ ID NOs: 30, 31, 32, 33, 34, 35 and 36. 21D4,21D4a, the amino acid sequence of V _K CDRl of 47G4,27F3,3C10,5G7,13F1 and 46E8, SEQ ID NO: shown in 37,38,39,40,41,42 and 43. 21D4,21D4a, the amino acid sequence of V _K CDR2 of 47G4,27F3,3C10,5G7,13F1 and 46E8, SEQ ID NO: shown in 44,45,46,47,48,49 and 50. 21D4,21D4a, the amino acid sequence of V _K CDR3 of 47G4,27F3,3C10,5G7,13F1 and 46E8, SEQ ID NO: shown in 51,52,53,54,55,56,57 and 58. CDR regions are illustrated using the Kabat system (Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services NIH Publication No. 91-3242).

Assuming that each of these antibodies binds to CD19 and antigen binding specificity is provided primarily by the CDR1, CDR2, and CDR3 regions, to generate other anti-CD19 binding molecules of the invention, V _H CDR1, CDR2, and CDR3 sequences and V _k CDR1, CDR2, and CDR3 sequences can be “mixed and matched” (ie, CDRs from different antibodies can be mixed and matched, The antibody must contain V _H CDR1, CDR2, and CDR3 sequences and V _k CDR1, CDR2, and CDR3 sequences). The CD19 binding of such “mixed matched” antibodies can be tested using the binding assays described above and in the examples (eg, ELISA, Biacore® analysis). it can. Preferably when mixed and matched V _H CDR sequences, the CDR1, CDR2, and / or CDR3 sequence from a particular V _H sequence is replaced with a structurally similar CDR sequence. Similarly, when mixing and matching V _k CDR sequences, CDR1, CDR2, and / or CDR3 from a particular V _k sequence is preferably replaced with a structurally similar CDR sequence. The sequence of one or more V _H and / or V _L CDR regions is structurally derived from the CDR sequences disclosed herein for monoclonal antibodies 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8. It will be readily apparent to one skilled in the art that new V _H and V _L sequences can be made by substituting similar sequences.

Accordingly, in another aspect, the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to CD19, preferably human CD19, comprising:
(A) SEQ ID NO: heavy chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of 16, 17, 18, 19, 20, 21, and 22;
(B) SEQ ID NO: heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of 23, 24, 25, 26, 27, 28 and 29;
(C) SEQ ID NO: Heavy chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of 30, 31, 32, 33, 34, 35 and 36;
(D) light chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39, 40, 41, 42 and 43;
(E) a light chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 45, 46, 47, 48, 49 and 50; and (f) SEQ ID NOs: 51, 52, 53 , 54, 55, 56, 57 and 58, a light chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of:
In a preferred embodiment, the antibody is:
(A) the heavy chain variable region CDR1 comprising SEQ ID NO: 16;
(B) heavy chain variable region CDR2 comprising SEQ ID NO: 23;
(C) heavy chain variable region CDR3 comprising SEQ ID NO: 30;
(D) a light chain variable region CDR1 comprising SEQ ID NO: 37;
(E) a light chain variable region CDR2 comprising SEQ ID NO: 44; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 51;
including.
In another preferred embodiment, the antibody is:
(A) the heavy chain variable region CDR1 comprising SEQ ID NO: 16;
(B) heavy chain variable region CDR2 comprising SEQ ID NO: 23;
(C) heavy chain variable region CDR3 comprising SEQ ID NO: 30;
(D) a light chain variable region CDR1 comprising SEQ ID NO: 37;
(E) a light chain variable region CDR2 comprising SEQ ID NO: 44; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 52;
including.
In another preferred embodiment, the antibody is:
(A) the heavy chain variable region CDR1 comprising SEQ ID NO: 17;
(B) heavy chain variable region CDR2 comprising SEQ ID NO: 24;
(C) heavy chain variable region CDR3 comprising SEQ ID NO: 31;
(D) a light chain variable region CDR1 comprising SEQ ID NO: 38;
(E) a light chain variable region CDR2 comprising SEQ ID NO: 45; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 53;
including.
In another preferred embodiment, the antibody is:
(A) the heavy chain variable region CDR1 comprising SEQ ID NO: 18;
(B) heavy chain variable region CDR2 comprising SEQ ID NO: 25;
(C) heavy chain variable region CDR3 comprising SEQ ID NO: 32;
(D) a light chain variable region CDR1 comprising SEQ ID NO: 39;
(E) a light chain variable region CDR2 comprising SEQ ID NO: 46; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 54;
including.
In another preferred embodiment, the antibody is:
(A) the heavy chain variable region CDR1 comprising SEQ ID NO: 19;
(B) heavy chain variable region CDR2 comprising SEQ ID NO: 26;
(C) heavy chain variable region CDR3 comprising SEQ ID NO: 33;
(D) a light chain variable region CDR1 comprising SEQ ID NO: 40;
(E) a light chain variable region CDR2 comprising SEQ ID NO: 47; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 55;
including.
In another preferred embodiment, the antibody is:
(A) the heavy chain variable region CDR1 comprising SEQ ID NO: 20;
(B) heavy chain variable region CDR2 comprising SEQ ID NO: 27;
(C) heavy chain variable region CDR3 comprising SEQ ID NO: 34;
(D) a light chain variable region CDR1 comprising SEQ ID NO: 41;
(E) a light chain variable region CDR2 comprising SEQ ID NO: 48; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 56;
including.
In another preferred embodiment, the antibody is:
(A) the heavy chain variable region CDR1 comprising SEQ ID NO: 21;
(B) heavy chain variable region CDR2 comprising SEQ ID NO: 28;
(C) heavy chain variable region CDR3 comprising SEQ ID NO: 35;
(D) a light chain variable region CDR1 comprising SEQ ID NO: 42;
(E) a light chain variable region CDR2 comprising SEQ ID NO: 49; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 57;
including.
In another preferred embodiment, the antibody is:
(A) the heavy chain variable region CDR1 comprising SEQ ID NO: 22;
(B) heavy chain variable region CDR2 comprising SEQ ID NO: 29;
(C) heavy chain variable region CDR3 comprising SEQ ID NO: 36;
(D) a light chain variable region CDR1 comprising SEQ ID NO: 43;
(E) a light chain variable region CDR2 comprising SEQ ID NO: 50; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 58;
including.

In the art, CDR3 domains can independently determine the binding specificity of antibodies to cognate antigens independently of CDR1 and / or CDR2 domains, and as expected, based on common CDR3 sequences It is well known that multiple antibodies with binding specificity can be generated. For example, Klimka et al., British J. of Cancer 83 (2) : 252-260 (2000) (describes the production of a humanized anti-CD30 antibody using only the heavy chain variable domain CDR3 of the mouse anti-CD30 antibody Ki-4. ); Beiboer et al., J. Mol. Biol. 296 : 833-849 (2000) (recombination using only the heavy chain CDR3 sequence of its parent mouse MOC-31 anti-epithelial glycoprotein-2 (EGP-2) antibody) Describes EGP-2 antibody); Rader et al., Proc. Natl. Acad. Sci. USA 95 : 8910-8915 (1998) (each member antibody contains a different sequence other than the CDR3 domain and its parent mouse) can bind to the same epitope as the parent murine antibody in antibody equal to or greater than the high affinity, humanized anti-integrin using a heavy and light chain variable CDR3 domain of murine anti-integrin alpha _V beta ₃ antibody LM609 alpha _V beta Describe a panel of ₃ antibodies); Barbas et al., J. Am. Chem. Soc. 116 : 2161-2162 (1994) (the CDR3 domain is responsible for its antigen binding) Barbas et al., Proc. Natl. Acad. Sci. USA 92 : 2529-2533 (1995) (Three Fabs for human placental DNA (SI-I, SI-40, And SI-32) describes replacing the existing heavy chain CDR3 by transplanting the heavy chain CDR3 sequence into the heavy chain of an anti-tetanus toxoid Fab and demonstrating that the CDR3 domain alone confers binding specificity Ditzel et al., J. Immunol. 157 : 739-749 (1996) (only the heavy chain CDR3 of the parent multispecific Fab LNA3 is transferred to the heavy chain of the monospecific IgG tetanus toxoid-binding Fab p313 antibody) To describe the grafting study that the binding specificity of its parent Fab was sufficiently maintained). Each of these references is hereby incorporated by reference in its entirety.

  Accordingly, the present invention provides a monoclonal antibody comprising one or more heavy and / or light chain CDR3 domains derived from an antibody derived from a human or non-human animal and capable of specifically binding to CD19. In certain aspects, the present invention provides monoclonal antibodies that comprise one or more heavy and / or light chain CDR3 domains from a non-human antibody, eg, a mouse or rat antibody, and that can specifically bind to CD19. To do. In some embodiments, such an antibody of the invention comprising one or more heavy and / or light chain CDR3 domains from a non-human antibody is (a) associated with a corresponding parent non-human antibody. Can compete; (b) retain functional characteristics; (c) bind to the same epitope; and / or (d) have similar binding affinities.

  In another aspect, the invention provides a human antibody capable of specifically binding to CD19, eg, a monoclonal antibody comprising one or more heavy and / or light chain CDR3 domains from a human antibody obtained from a non-human animal. I will provide a. In other aspects, the present invention provides one or more heavy and / or light chain CDR3 domains from a first human antibody capable of specifically binding to CD19, eg, a human antibody obtained from a non-human animal. A monoclonal antibody wherein the CDR3 domain from the first human antibody is replaced with the CDR3 domain of a human antibody lacking binding specificity for CD19 to yield a first human antibody that can specifically bind to CD19. provide. In some embodiments, such an antibody of the invention comprising one or more heavy and / or light chain CDR3 domains from a first human antibody comprises a corresponding parent first human antibody (a ) Can compete for binding; (b) retain functional characteristics; (c) bind to the same epitope; and / or (d) have similar binding affinity.

Antibodies with specific germline sequences In certain embodiments, the antibodies of the present invention are derived from heavy chain variable regions from specific germline heavy chain immunoglobulin genes and / or from specific germline light chain immunoglobulin genes. Of the light chain variable region.

For example, in a preferred embodiment, the present invention provides an isolated monoclonal antibody or antigen thereof that specifically binds to CD19, comprising a heavy chain variable region that is the product of or derived from a human V _H 5-51 gene. Provide a binding part. In another preferred embodiment, the present invention provides an isolated monoclonal antibody or antigen thereof that specifically binds CD19, comprising a heavy chain variable region that is the product of or derived from a human V _H 1-69 gene. Provide a binding part. In yet another preferred embodiment, the invention comprises a light chain variable region or derived from this is the product of a human V _K L18 gene, specifically monoclonal antibodies or antigen binding isolated that bind to CD19 Provide part. In yet another preferred embodiment, the invention comprises a light chain variable region or derived from this is the product of a human V _K A27 gene, specifically monoclonal antibodies or antigen binding isolated that bind to CD19 Provide part. In yet another preferred embodiment, the invention comprises a light chain variable region or derived from this is the product of a human V _K L15 gene, specifically monoclonal antibodies or antigen binding isolated that bind to CD19 Provide part. In yet another preferred embodiment, the present invention provides:
(A) Heavy chain variable that is the product of or derived from the human _VH 5-51 or 1-69 genes, which encode the amino acid sequences shown in SEQ ID NOs: 74 and 75, respectively. Including areas;
(B) the product of or derived from the human V _K L18, V _K A27 or V _K L15 gene (these genes encode the amino acid sequences shown in SEQ ID NO: 76, 77 and 78, respectively) And (c) specifically binds to CD19, preferably human CD19,
An isolated monoclonal antibody or antigen-binding portion thereof is provided.

Examples of antibodies having V _H and V _K of V _H 5-51 and V _K L18, respectively, are 21D4, 21D4a, 27F3, 5G7, 13F1 and 46E8. An example of an antibody having V _H 1-69 and V _K A27, V _H and V _K , respectively, is 47G4. An example of an antibody having V _H 1-69 and V _K L15, V _H and V _K , respectively, is 3C10.

  As used herein, a human antibody is a heavy product that is “product of” or “derived from” a particular germline sequence when the variable region of the antibody is obtained from a system that uses human germline immunoglobulin genes. It includes a chain or light chain variable region. Such systems include immunizing a transgenic mouse having a human immunoglobulin gene with the antigen of interest, or screening a human immunoglobulin gene library shown in phage with the antigen of interest. A human antibody that is “product of” or “derived from” a human germline immunoglobulin sequence is a step that compares the amino acid sequence of the human antibody with the amino acid sequence of the human germline immunoglobulin, and a sequence relative to the sequence of the human antibody. Can be identified by selecting the human germline immunoglobulin sequence that is closest (ie, with the greatest% identity). A human antibody that is “product of” or “derived from” a particular human germline immunoglobulin sequence may contain a germline sequence, eg, for the deliberate introduction of naturally occurring somatic mutations or site-specific mutations. In comparison, amino acid differences may be included. However, the selected human antibody is typically at least 90% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene and is reproductive from other species (eg, mouse germline sequence). Contains amino acid residues that identify a human antibody as being human, as compared to a series immunoglobulin amino acid sequence. In certain cases, human antibodies may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Good. Typically, human antibodies derived from a particular human germline sequence will only show a difference of only 10 amino acids from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, human antibodies may only show a difference of only 5 amino acids, or even 4, 3, 2, or 1 amino acid sequences encoded by germline immunoglobulin genes.

Homologous antibodies In yet another embodiment, the antibodies of the invention comprise heavy and light chain regions comprising amino acid sequences that are homologous to the amino acid sequences of preferred antibodies described herein, and anti-CD19 antibodies of the invention It retains the desired functional properties.

For example, the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof comprising a heavy chain variable region and a light chain variable region as follows:
(A) the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7;
(B) the light chain variable region comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15;
(C) the antibody binds with K _D 1 × 10 ^-7 M or less for human CD19;
(D) Binds to Raji B cell tumor cells and Daudi B cell tumor cell antibodies.
In various embodiments, the antibody can be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

In other embodiments, the _VH and / or _VL amino acid sequences may be 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the sequences described above. Good. Antibodies having V _H and V _L region having V _H and V _L region and a high (i.e. 80% or higher) homology above sequences, SEQ ID NO: 59,60,61,62,63, The functionalities described herein after mutagenesis (eg, site-specific or PCR mutagenesis) of nucleic acid molecules encoding 64, 65, 66, 67, 68, 69, 70, 71, 72 and 73 The assay can be used to test for the retained function of the encoded altered antibody (ie, the functions described in (c)-(d) above).

  As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between two sequences is the number of gaps that need to be introduced for optimal alignment of the two sequences, the number of identical positions shared by the sequences, taking into account the length of each gap. Function (ie,% homology = number of identical positions / total number of positions × 100). Comparison of sequences between two sequences and determination of percent identity can be performed using a mathematical algorithm as described in the following non-limiting examples.

  The percent identity between two amino acid sequences is determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci. 4: 11-17 (1988)) incorporated into the ALIGN program (version 2.0). , PAM120 weighted residue table, gap length penalty 12 and gap penalty 4 can be used. In addition, the percent identity between two amino acid sequences is determined by Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (J. Mol. Biol. 48: 444-453) incorporated in the GAP program in the GCG software package (available at www.gcg.com). 1970)), gap weight 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4 using either Blossum 62 matrix or PAM250 matrix , 5, or 6 can be used.

  Additionally or alternatively, the protein sequences of the present invention can further be used as “query sequences” to search public databases, eg, to identify related sequences. Such a search can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215: 403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences that are homologous to the antibody molecules of the present invention. Gapped BLAST as described in Altschul et al. (1997) Nucleic Acids Res. 25 (17): 3389-3402 can be used to obtain a gaped alignment for comparison purposes. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

Antibodies with Conservative Modifications In certain embodiments, the antibodies of the invention comprise a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein these CDRs One or more of the sequences comprises a preferred antibody described herein (eg, 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 or 46E8) or a specific amino acid sequence based on conservative modifications thereof, Retains the desired functional properties of the anti-CD19 antibodies of the present invention. Accordingly, the present invention provides an isolated monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, which are: I will provide a.
(A) the heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 30, 31, 32, 33, 34, 35 and 36 and conservative modifications thereof;
(B) the light chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57 and 58 and conservative modifications thereof;
(C) the antibody binds to CD19 with a K _{D of} 1 × 10 ⁻⁷ M or less;
(D) Binds to Raji B cell tumor cells and Daudi B cell tumor cells.

  In a preferred embodiment, the heavy chain variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 23, 24, 25, 26, 27, 28, and 29, and conservative modifications thereof; And the light chain variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 44, 45, 46, 47, 48, 49 and 50, and conservative modifications thereof. In another preferred embodiment, the heavy chain variable region CDR1 sequence is an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 16, 17, 18, 19, 20, 21, and 22, and conservative modifications thereof. And the light chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 37, 38, 39, 40, 41, 42 and 43, and conservative modifications thereof.

  In various embodiments, the antibody can be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

  As used herein, “conservative sequence modifications” are intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody comprising an amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. A family of amino acid residues having similar side chains has been defined in the art. These families include basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, Tyrosine, cysteine, tryptophan), nonpolar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (eg threonine, valine, isoleucine), and aromatic side chains ( For example, amino acids having tyrosine, phenylalanine, tryptophan, histidine) are included. Thus, one or more amino acid residues in the CDR regions of the antibodies of the invention can be replaced with other amino acids of the same side chain family, and the altered antibody can be functionalized as described herein. Can be tested for retained function (i.e., functions described in (c)-(f) above).

Antibodies that bind to the same epitope as an anti-CD19 antibody of the invention In another embodiment, the invention relates to any CD19 monoclonal antibody of the invention (ie, any monoclonal antibody of the invention for binding to CD19) in human CD19. An antibody that binds to the same epitope as the antibody having the ability to cross-compete). In a preferred embodiment, the reference antibody for cross-competition studies is monoclonal antibody 21D4 (having the V _H and _VL sequences shown in SEQ ID NOs: 1 and 8, respectively), or monoclonal antibody 21D4a (SEQ ID NO: 1 and shown in 9, respectively, V _H sequences and V _L having the sequence), or the monoclonal antibody 47G4 (SEQ ID NO: shown in 2 and 10, respectively, having the V _H and V _L sequences), or the monoclonal antibody 27F3 ( SEQ ID NOs: 3 and 11, with V _H and _VL sequences, respectively, or monoclonal antibody 3C10 (shown with SEQ ID NOs: 4 and 12, respectively, with V _H and _VL sequences) or Monoclonal antibody 5G7 (shown in SEQ ID NOs: 5 and 13, with _VH and _VL sequences, respectively) or monoclonal antibody 13F1 (shown in SEQ ID NOs: 6 and 14, respectively, _VH and _VL sequences, respectively) Or monoclonal antibody 46E8 (shown in SEQ ID NOs: 7 and 15, respectively, with _VH and _VL sequences). Such cross-competing antibodies can be identified based on their ability to cross-compete with 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 or 46E8 in standard CD19 binding assays. For example, BIAcore® analysis, ELISA method, or flow cytometry may be used to demonstrate the ability to cross-compete with the antibodies of the invention. For example, the ability of a test antibody to inhibit the binding of 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 or 46E8 to human CD19 indicates that the test antibody is capable of inhibiting 21D4, 21D4a, 47G4, 27F3, 3C10, It can compete with 5G7, 13F1 or 46E8 and thus proves to bind to the same epitope in human CD19 as 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 or 46E8. In a preferred embodiment, the antibody that binds to the same epitope in human CD19 as 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 or 46E8 is a human monoclonal antibody. Such human monoclonal antibodies can be prepared and isolated as described in the examples.

Engineered and modified antibodies Antibodies having one or more of the V _H and / or _VL sequences disclosed herein can be used as starting materials for engineering modified antibodies, the modified antibodies being starting antibodies May have altered properties when compared to. An antibody can be one or both variable regions (ie, V _H and / or V _L ), eg, one or more CDR regions, and / or one or more framework regions It can be manipulated by modifying multiple amino acids. Additionally or alternatively, antibodies can be manipulated, for example, by altering residues within the constant region to alter the effector functions of the antibody.

  In certain embodiments, CDR grafting can be used to manipulate the variable region of an antibody. Antibodies primarily interact with target antigens through amino acid residues present in six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Since CDR sequences are involved in most antibody-antigen interactions, construct expression vectors containing CDR sequences from specific naturally occurring antibodies grafted into framework sequences from different antibodies with different properties By doing so, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies (eg, Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P (1986) Nature 321: 522-525; Queen, C. et al. (1989) Proc. Natl. Acad. See. USA 86: 10029-10033; US Pat. No. 5,225,539 to Winter, and US Pat. No. 5,530,101; 5,585,089; 5,693,762; and 6,180,370).

Thus, another embodiment is SEQ ID NO: 16, 17, 18, 19, 20, 21 and 22, SEQ ID NO: 23, 24, 25, 26, 27, 28 and 29, and SEQ ID NO: 30 A heavy chain variable region comprising a CDR1, CDR2, and CDR3 sequence comprising an amino acid sequence selected from the group consisting of: 31, 32, 33, 34, 35 and 36; and SEQ ID NOs: 37, 38, 39, 40, 41, 42, and 43 and SEQ ID NOs: 44, 45, 46, 47, 48, 49 and 50, and SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57 and 58 and 12 The present invention relates to an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a light chain variable region comprising CDR1, CDR2, and CDR3 sequences each comprising an amino acid sequence selected from each other. Thus, such antibodies comprise the V _H and V _L CDR sequences of monoclonal antibodies 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 or 46E8, but still contain the different framework regions of these antibodies. But you can.

Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences for the human heavy and light chain variable region genes, along with the “VBase” human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase) Kabat, EA et al. (1991) “Sequences of Proteins of Immunological Interest”, 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM, the contents of which are specifically incorporated herein by reference. (1992) "The Repertoire of Human Germline V _H Sequences Reveals about Fifty Groups of V _H Segments with Different Hypervariable Loops", J. Mol. Biol. 227: 776-798; and Cox, JPL et al. (1994) "A Directory of Human Germ-line V _H Segments Reveals a Strong Bias in their Usage. ”Eur. J. Immuol. 24: 827-836. As another example, germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain germline sequences found in HCo7 HuMAb mice are available with the Genbank accession numbers shown here: 1-69 (NG_0010109, NT_024637, and BC070333), 3-33 (NG_0010109 and NT_024637) , And 3-7 (NG_0010109 and NT_024637). As another example, the following heavy chain germline sequences found in HCo12 HuMAb mice are available with the Genbank accession numbers shown here: 1-69 (NG_0010109, NT_024637, and BC070333), 5-51 (NG_0010109) And NT_024637), 4-34 (NG_0010109 and NT_024637), 3-30.3 (CAJ556644), and 3-23 (AJ406678).

The protein sequence of the antibody is a protein sequence database compiled using one of the sequence similarity search methods known as Gapped BLAST (Altsuchul et al. (1997) Nuleic Acids Research 25 : 3389-3402) Compared with BLAST is a heuristic algorithm in that a statistically significant alignment between antibody and database sequences can include high-scoring segment pairs (HSP) of aligned letters. A segment pair whose score cannot be improved by stretching or shortening is called a hit. Briefly, the nucleotide sequence from VBASE (http://vbase.mrc-cpe.cam.ac.uk/vbase1/list2.php) is translated and includes the FR1 to FR3 framework region and the region in between The area is preserved. The average length of the database sequence is 98 residues. Duplicate sequences that exactly match the full length of the protein are removed. BLAST searches of proteins using the blastp program with default standard parameters except for turning off the low complexity filter and the substitution matrix of BLOSUM62, the top 5 hits that produce sequence matches Filter. The nucleotide sequence is translated in all six frames, and frames without stop codons in the matched database sequence segments are considered potential hits. This is then confirmed using the BLAST program tblastx which translates the antibody sequence of all six frames and compares the translation to the dynamically translated VBASE nucleotide sequence in all six frames.

  Identity is an exact amino acid match between the antibody sequence and the protein database that spans the entire length of the sequence. A positive (identical + substitution match) is an amino acid substitution that is not identical but is derived by a BLOSUM62 substitution matrix. If the antibody sequence matches two of the database sequences with the same identity, the highest positive hit can be determined to be the hit of the matching sequence.

Preferred framework sequences for use in the antibodies of the invention are structurally similar to the framework sequences used by selected antibodies of the invention, eg, the V _H 5-51 frame used by preferred monoclonal antibodies of the invention. Work sequence (SEQ ID NO: 74) and / or V _H 1-69 framework sequence (SEQ ID NO: 75) and / or V _K L18 framework sequence (SEQ ID NO: 76) and / or V _K A27 frame A sequence similar to the work sequence (SEQ ID NO: 77) and / or the V _K L15 framework sequence (SEQ ID NO: 78). V _H CDR1, CDR2, and CDR3 sequences and V _K CDR1, CDR2, and CDR3 sequences are grafted into a framework region that has the same sequence as that found in the germline immunoglobulin gene from which the framework sequence is derived Or the CDR sequences can be transplanted into framework regions containing one or more mutations compared to the germline sequence. For example, it may be useful to mutate residues in the framework regions to maintain or enhance the antigen-binding ability of the antibody in certain cases (eg, US Pat. Nos. 5,530,101; Queen, et al., 5,585,089 to Queen; No. 5,693,762; and 6,180,370).

Another type of variable region mutation mutates the amino acid sequence in the V _H and / or V _K CDR1, CDR2, and / or CDR3 regions, thereby causing one or more binding properties (eg, (Affinity) is improved. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations and its effects on antibody binding or other functional properties of interest are described and practiced herein. It can be evaluated in the in vitro or in vivo assays provided in the examples. Preferably conservative modifications (as described above) are introduced. The mutation may be an amino acid substitution, addition, or deletion, but is preferably a substitution. Moreover, typically only 1, 2, 3, 4, or 5 residues are changed within the CDR regions.

Accordingly, in another embodiment, the disclosure provides: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 20, 21 and 22, or SEQ ID NOs: 16, 17 , V _H CDRl region comprising an amino acid sequence having amino acid substitutions, deletions, or additions four or five when compared with 18, 19, 20, 21 and 22; (b) SEQ ID NO : An amino acid sequence selected from the group consisting of 23, 24, 25, 26, 27, 28 and 29, or amino acid substitutions when compared to SEQ ID NO: 23, 24, 25, 26, 27, 28 and 29; V _H CDR2 region comprising an amino acid sequence having 1, 2, 3, 4 or 5 deletions or additions; (c) from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, 35 and 36 Amino acid sequence selected, or amino acid substitutions, deletions, or additions 1, 2, 3, 4 or 5 when compared to SEQ ID NO: 30, 31, 32, 33, 34, 35 and 36 V _H CDR3 region comprising an amino acid sequence having a number; (d) SEQ ID NO: 37,38,39,40,41,42 and amino acid sequence selected from the group consisting of 43, or SEQ ID NO: 37, 38 V _K CDR1 region comprising an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions when compared to 39, 40, 41, 42 and 43; (e) SEQ ID NO : An amino acid sequence selected from the group consisting of 44, 45, 46, 47, 48, 49 and 50, or amino acid substitutions when compared to SEQ ID NO: 44, 45, 46, 47, 48, 49 and 50; V _K CDR2 region comprising amino acid sequence having 1, 2, 3, 4 or 5 deletions or additions; (f) consisting of SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57 and 58 An amino acid sequence selected from the group, or amino acid substitutions, deletions or additions 1, 2, 3, 4 or 5 when compared to SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57 and 58 Including, V _K CDR3 region comprising an amino acid sequence having a number, comprising a heavy chain variable region, provides an anti-CD19 monoclonal antibodies isolated or antigen binding portion thereof.

Engineered antibodies of the invention include antibodies that have been modified to framework residues within V _H and / or V _K , for example, to improve the properties of the antibody. Typically, such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to “backmutate” one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived.

  For example, Table 1 below shows a number of amino acid changes in the framework regions of anti-PD-1 antibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 that differ from the parental germline sequence of their heavy chain. In order to return one or more of the amino acid residues in the framework region sequence to their germline configuration, somatic variants may be transformed into their germline by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. It can be “backmutated” into the sequence.

(Table 1) Modification of heavy chain germline composition to antibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4

  Another type of framework modification is one in the framework region or in one or more CDR regions to remove T cell epitopes, thereby reducing the possible immunogenicity of the antibody. Mutating one or more residues. This approach is also described in further detail in US Patent Publication No. 20030153043 by Carr et al.

  In addition to or instead of modifications made within the framework or CDR regions, the antibodies of the invention typically have serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity. In order to alter one or more functional properties of an antibody such as, it may be engineered to include modifications within the Fc region. Furthermore, an antibody of the invention may be chemically modified (eg, one or more chemical moieties can be attached to the antibody) or again to alter its glycosylation so as to alter its glycosylation. Modifications may be made to change the functional properties. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is as per Kabat's EU index.

  In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region changes, eg, increases or decreases. This approach is further described in US Pat. No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CH1 is altered, for example, to facilitate assembly of light and heavy chains, or to increase or decrease antibody stability.

  In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half life of the antibody. More specifically, one or more amino acid mutations are made in the CH2-CH3 of the Fc-hinge fragment so that the antibody has impaired staphylococcal protein A (SpA) binding compared to native Fc-hinge domain SpA binding. Introduce into the domain interface region. This approach is described in further detail in US Pat. No. 6,165,745 by Ward et al.

  In another embodiment, the antibody is modified to increase its biological half life. Various approaches are possible. For example, one or more of the following mutations can be introduced as described in US Pat. No. 6,277,375 to Ward et al. T252L, T254S, T256F. Alternatively, to increase biological half-life, salvage receptors derived from two loops of the CH2 domain of the Fc region of IgG as described by Presta et al. In US Pat. Nos. 5,869,046 and 6,121,022. Antibodies can be altered within the CH1 or CL region to include a binding epitope.

  In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 may have a modified affinity for an effector ligand but the parent antibody The amino acid residues can be substituted with different amino acid residues so as to retain the antigen-binding ability. An effector ligand with altered affinity for it can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in US Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

  In another example, the antibody is selected from amino acid residues 329, 331, and 322 to exhibit altered C1q binding and / or decreased or eliminated complement dependent cytotoxicity (CDC). One or more amino acids can be substituted with different amino acid residues. This approach is described in further detail in US Pat. No. 6,194,551 by Idusogie et al.

  In another example, one or more amino acid residues within amino acids 2316, 17, 18, 19, 20, 21, and 2239 are changed, thereby changing the ability of the antibody to fix complement. This approach is described in detail in PCT Publication No. 94/29351 by Bodmer et al.

  In yet another example, the Fc region has the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280 , 283,285,286,289,290,292,293,294,295,296,298,301,303,305,307,309,312,315,320,322,324,326,327,329,330 , 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439 To increase the antibody's ability to mediate antibody-dependent cytotoxicity (ADCC) and / or to increase the affinity of the antibody for the Fcγ receptor by modifying one or more amino acids of Modified. This approach is described in detail in Presta PCT Gazette WO 00/42072. In addition, binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn have been mapped and variants showing improved binding have been described (Shields, RL et al. (2001), J. Biol. Chem. 276: 6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII. In addition, the following complex mutants have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A and S298A / E333A / K334A.

  In yet another embodiment, the glycosylation of the antibody is altered. For example, an aglycosylated antibody can be made (ie, the antibody is deficient in glycosylation). Glycosylation can be altered to increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be made, for example, by changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for the antigen. Such an approach is described in further detail in US Pat. Nos. 5,714,350 and 6,350,861 to Co et al.

Additionally or alternatively, antibodies with altered types of glycosylation can be made, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structure . Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be made, for example, by expressing the antibody in a host cell that has an altered glycosylation mechanism. Cells with altered glycosylation mechanisms have been described in the art and can be used as host cells to express the recombinant antibodies of the invention and thereby produce antibodies with altered glycosylation. it can. For example, the cell lines Ms704, Ms705, and Ms709 have the fucosyltransferase gene FUT8 (α (1,6) so that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose in their hydrocarbons. ) Fucosyltransferase). The Ms704, Ms705, and Ms709 FUT8 ^{− / −} cell lines were generated by targeted disruption of the FUT8 gene of CHO / DG44 cells using two replacement vectors (US Patent Publication No. 20040110704 and Yamane by Yamane et al. -See Ohnuki et al. (2004) Biotechnol Bioeng 87: 614-22). As another example, European Patent No. 1,176,195 by Hanai et al. Shows that antibodies expressed in such cell lines show hypofucosylation by reducing or eliminating α1,6 binding-related enzymes. A cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase has been described. Hanai et al. Also have cell lines that have low or no enzymatic activity to add fucose to N-acetylglucosamine that binds to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 ( ATCC CRL 1662). PCT Gazette No. 03/035835 by Presta describes Lec 13 cells, a variant CHO cell line with reduced ability to bind fucose to Asn (297) -linked hydrocarbons, as well as their host cells. (See also Shields, RL et al. (2002), J. Biol. Chem. 277: 26733-26740). PCT Publication No. 99/54342 by Umana et al. Shows that antibodies expressed in engineered cell lines show an increase in the bisecting GlcNac structure, which results in an increase in the ADCC activity of the antibody (as well , Umana et al. (1999) Nat. Biotech. 17: 176-180), glycoprotein-modified glycosyltransferases (eg, β (1,4) -N-acetylglucosaminyltransferase III (GnT III)). A cell line engineered to express). Alternatively, the fucose residue of the antibody may be cleaved using a fucosidase enzyme. For example, the fucosidase α-L-fucosidase removes fucosyl residues from antibodies (Tarentino, AL et al. (1975) Biochem. 14: 5516-23).

  Another modification of the antibodies described herein contemplated by the present invention is PEGylation. An antibody can be PEGylated, for example, to increase the biological (eg, serum) half life of the antibody. To PEGylate an antibody, the antibody or fragment thereof is typically a polyethylene such as a reactive ester or aldehyde derivative of PEG, provided that one or more PEG groups become attached to the antibody or antibody fragment. React with glycol (PEG). Preferably, PEGylation is performed by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is used to derivatize other proteins, such as mono (C1-C10) alkoxy or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. Is intended to include any type of PEG. In certain embodiments, the PEGylated antibody is an aglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the present invention. See, for example, European Patent No. 0 154 316 by Nishimura et al. And European Patent No. 0 401 384 by Ishikawa et al.

Antibody Physical Properties The antibodies of the present invention can be further characterized by various physical properties of anti-CD19 antibodies. Various assays can be used to detect and / or distinguish different antibody classes based on these physical properties.

In some embodiments, the antibodies of the invention may comprise one or more glycosylation sites in either the light chain variable region or the heavy chain variable region. The presence of one or more glycosylation sites in the variable region increases the immunogenicity of the antibody and changes in the antibody's pK due to changes in antigen binding (Marshall et al (1972) Annu Rev Biochem 41 : 673-702; Gala FA and Morrison SL (2004) J Immunol 172 : 5489-94; Wallick et al (1988) J Exp Med 168 : 1099-109; Spiro RG (2002) Glycobiology 12 : 43R-56R; Parekh et al (1985) Nature 316 : 452-7; Mimura et al. (2000) Mol Immunol 37 : 697-706). Glycosylation is known to occur at motifs containing NXS / T sequences. Variable region glycosylation is tested using the Glycoblot assay, which cleaves the antibody to produce Fab and then tests glycosylation using an assay that measures periodate oxidation and Schiff base formation. Can be done. Alternatively, glycosylation of variable regions can be tested using Dionex light chromatography (Dionex-LC), which cleaves saccharides from the Fab into monosaccharides and analyzes the content of individual saccharides. In some instances, it is preferred that the anti-CD19 antibody does not contain variable region glycosylation. This can be accomplished either by selecting antibodies that do not contain a glycosylation motif in the variable region or by mutating residues within the glycosylation motif using standard techniques well known to those skilled in the art. .

  In a preferred embodiment, the antibodies of the invention do not contain asparagine isomerism sites. Deamidation or isoaspartic acid action can occur on NG or DG sequences, respectively. Deamidation or isoaspartic acid action forms isoaspartic acid which reduces the stability of the antibody by forming a twisted structure from the carboxy terminus of the side chain rather than the main chain. The production of isoaspartic acid can be measured using an iso-quant assay that tests for isoaspartic acid using reverse phase HPLC.

Each antibody may have a unique isoelectric point (pI), but generally antibodies are included in the pH range of 6-9.5. The pI of IgG1 antibody is typically included in the pH range of 7-9.5, and the pI of IgG4 antibody is typically included in the pH range of 6-8. The antibody may have a pI outside this range. Although its effect is not generally known, there is the theory that antibodies with a pI outside the normal range may unfold to some extent under in vivo conditions and may be unstable. The isoelectric point can be tested using a capillary isoelectric focusing assay that forms a pH gradient and can utilize laser focusing to improve accuracy (Janini et al (2002) Electrophoresis 23 : 1605-11; Ma et al. (2001) Chromatographia 53 : S75-89; Hunt et al (1998) J Chromatogr A 800 : 355-67). In some instances, it is preferred that the anti-CD19 antibody contains a pI value that falls within the normal range. This can be achieved either by screening for antibodies with a pI within the normal range or by mutating charged surface residues using standard techniques well known in the art.

Each antibody may have a melting temperature indicative of thermal stability (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3 : 361-71). The high thermal stability indicates the high stability of the whole antibody in vivo. The melting point of an antibody can be measured using techniques such as differential scanning calorimetry (Chen et al (2003) Pharm Res 20 : 1952-60; Ghirlando et al (1999) Immunol Lett 68 : 47-52). T _M1 indicates the temperature of the initial unfolding of the antibody. T _M2 indicates the temperature of complete unfolding of the antibody. In general, the _TM1 of antibodies of the present invention is greater than 60 ° C, preferably greater than 65 ° C, and even more preferably greater than 70 ° C. Alternatively, the thermal stability of an antibody can be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40 : 343-9).

In a preferred embodiment, antibodies are selected that do not degrade rapidly. Fragmentation of anti-CD19 antibodies can be measured using capillary electrophoresis (CE) and MALDI-MS, which are well understood in the art (Alexander AJ and Hughes DE (1995) Anal Chem 67 : 3626-32).

  In another preferred embodiment, antibodies are selected that have minimal aggregation. Aggregation can result in undesirable immune response factors and / or altered or undesirable pharmacological properties. Generally 25% or less, preferably 20% or less, even more preferably 15% or less, even more preferably 10% or less, even more preferably 5% or less Antibodies with are acceptable. Aggregation is measured by several techniques well known in the art including size exclusion column (SEC) high performance liquid chromatography (HPLC) and light scattering to identify monomers, dimers, trimers, or multimers Can be done.

Methods for Manipulating Antibodies As described above, anti-CD19 antibodies having V _H and V _K sequences disclosed herein are used to alter VH and / or V _K sequences, or constant regions bound thereto. Thus, a novel anti-CD19 antibody can be produced. Thus, in another aspect of the invention, the structural features of the anti-CD19 antibodies of the invention, such as 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 or 46E8 are used to bind to human CD19. A structurally related anti-CD19 antibody is produced that retains at least one functional property of the antibody of the invention. For example, one or more CDR regions of 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 or 46E8 or variants thereof may be combined with known framework regions and / or other CDRs by recombination, As described, further recombinantly produced anti-CD19 antibodies of the invention can be made. Other types of modifications include those described in the previous chapter. The starting material for the engineered method is one or more of the V _H and / or V _K sequences provided herein or one or more CDR regions thereof. In order to make engineered antibodies, there is actually a need to prepare antibodies having one or more of the V _H and / or V _K sequences provided herein, or one or more CDR regions thereof. Absent. Rather, the information contained in the sequence is used as starting material to create a “second generation” sequence derived from the original sequence, and then the “second generation” sequence is prepared and expressed as a protein.

Accordingly, in another embodiment, the present invention provides a method of preparing an anti-CD19 antibody comprising:
(A) (i) SEQ ID NOs: 16, 17, 18, 19, 20, 21 and 22 CDR1 sequences selected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, 27, 28 and 29 A heavy chain variable region antibody sequence comprising a CDR2 sequence selected from the group consisting of and / or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, 35 and 36; and / Or (ii) CDR1 sequences selected from the group consisting of SEQ ID NOs: 37, 38, 39, 40, 41, 42 and 43, SEQ ID NOs: 44, 45, 46, 47, 48, 49 and 50 A light chain variable region antibody sequence comprising a CDR2 sequence selected from the group consisting of and / or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57 and 58, Providing a stage;
(B) changing at least one amino acid residue in the heavy chain variable region antibody sequence and / or the light chain variable region antibody sequence to produce at least one altered antibody sequence; and (c) the altered antibody Expressing the sequence as a protein.

  The altered antibody sequences can be prepared and expressed using standard molecular biology techniques.

Preferably, the antibody encoded by the altered antibody sequence has one, some, or all of the functional properties of an anti-CD19 antibody described herein, including, but not limited to, functional properties It is an antibody to be retained.
(I) binds to human CD19 with a K _{D of} 1 × 10 ⁻⁷ M or less;
(Ii) binds to Raji B cell tumor cells and Daudi B cell tumor cells.

  The functional properties of the modified antibodies can be determined using standard assays (eg, flow cytometry, binding assays) that can be used in the art and / or described herein, as described in the Examples. Can be used to evaluate.

  In certain embodiments of the methods of manipulating the antibodies of the present invention, the resulting modified anti-CD19 antibody can be randomly or selectively introduced with mutations along all or part of the anti-CD19 antibody coding sequence. Can be screened for binding activity and / or other functional properties as described herein. Mutation methods have been described in the art. For example, PCT Publication WO 02/092780 by Short describes a method for producing and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, PCT publication WO 03/074679 by Lazar et al. Describes a method of using a computer screening method to optimize the physicochemical properties of an antibody.

Nucleic acid molecules encoding antibodies Another aspect of the invention pertains to nucleic acid molecules encoding the antibodies of the invention. The nucleic acid may be present in whole cells, cell lysates, or partially purified or substantially purified forms. Nucleic acids are purified from other cellular components or other contaminants by standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and other techniques well known in the art. “Isolated” or “substantially pure”. See, for example, F. Ausubel et al. (1987) “Current Protocols in Molecular Biology”, Greene Publishing and Wiley Interscience, New York. The nucleic acid of the present invention can be, for example, DNA or RNA and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

  The nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by a hybridoma (eg, a hybridoma prepared from a transgenic mouse having a human immunoglobulin gene, as described further below), the cDNA encoding the light and heavy chains of the antibody produced by the hybridoma is Can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (eg, using phage display technology), one or more nucleic acids encoding the antibody can be recovered from the library.

  Preferred nucleic acid molecules of the invention are molecules that encode the VH and VL sequences of 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 or 46E8 monoclonal antibodies. The DNA sequences encoding the VH sequences of 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8 are shown in SEQ ID NOs: 59, 60, 61, 62, 63, 64 and 65, respectively. The DNA sequences encoding the VL sequences of 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8 are shown in SEQ ID NOs: 66, 67, 68, 69, 70, 71, 72 and 73, respectively.

After DNA fragments encoding the _VH and _VL segments are obtained, these DNA fragments are standardized, for example, to convert variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. Can be further manipulated by simple recombinant DNA techniques. In these manipulations, a _VL or _VH coding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term “functionally linked” is intended to mean joining two DNA fragments such that the amino acid sequence encoded by the two DNA fragments still remains in-frame. Is done.

The isolated DNA encoding the VH region, a V _H coding DNA, by operably linked to another DNA molecule encoding heavy chain constant regions (CH1, CH2, and CH3), the heavy chain of the full length It can be converted into a gene. The sequences of human heavy chain constant region genes are known in the art (eg, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH Publication No. 91- 3242), DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but most preferably is an IgG1 or IgG4 constant region. For Fab fragment heavy chain genes, VH-encoding DNA can be operably linked to another DNA molecule that encodes only the heavy chain CH1 constant region.

The isolated DNA encoding the V _L region, the VL encoding DNA, by operatively linked to another DNA molecule encoding the light chain constant region CL, be converted into light chain gene of full-length Can do. The sequence of the human light chain constant region gene is known in the art (eg, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242). DNA fragments containing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region can be a kappa or lambda constant region.

To generate the scFv gene, V _H and V _L coding DNA fragments so that the V _H and V _L sequences can be expressed as a continuous single chain protein with the V _L and V _H regions joined by a flexible linker Is operably linked to a flexible linker, eg, another fragment encoding the amino acid sequence (Gly ₄ -Ser) ₃ (eg, Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc Natl. Acad. Sci. USA 85: 5879-5883; see McCafferty et al. (1990) Nature 348: 552-554).

Production of Monoclonal Antibodies Monoclonal antibodies (mAbs) of the present invention are produced by a variety of techniques, including standard monoclonal antibody methodologies, such as the standard somatic cell hybridization technique of Kohler and Milstein (1975) Nature 256: 495. be able to. Somatic cell hybridization techniques are preferred in principle, but other techniques for producing monoclonal antibodies can be used, such as viral or oncogene transformation of B lymphocytes.

  A preferred animal system for preparing hybridomas is the murine system. Hybridoma production in mice is a very well established technique. Immunization protocols and techniques for isolating immunized spleens for fusion are known in the art. Fusion partners (eg, mouse myeloma cells) and fusion techniques are also known.

  The chimeric or humanized antibody of the present invention can be prepared based on the sequence of the non-human monoclonal antibody prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the subject mouse hybridoma and engineered to include non-mouse (eg, human) immunoglobulin sequences using standard molecular biology techniques. Can do. For example, to make chimeric antibodies, mouse variable regions can be linked to human constant regions using methods known in the art (see, eg, US Pat. No. 4,816,567 to Cabilly et al.). To generate humanized antibodies, mouse CDR regions can be inserted into the human framework using methods known in the art (eg, Winter et al. US Pat. No. 5,225,539, and Queen et al. US Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370).

  In a preferred embodiment, the antibody of the present invention is a human monoclonal antibody. Such human monoclonal antibodies against CD19 can be generated using transgenic or transchromosomal mice that have part of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include mice referred to herein as HuMab mice (registered trademark) and KM mice (registered trademark), respectively, which are collectively referred to herein as "human Ig mice. "

  HuMab mouse® (Medarex®, Inc.) is a non-rearranged human heavy (μ and γ) and κ light chain immunity with targeted mutations that inactivate endogenous μ and κ chain loci. It contains a human immunoglobulin gene minilocus that encodes a globulin sequence (eg, Lonberg et al. (1994) Nature 368 (6474): 856-859). Thus, mice show decreased expression of mouse IgM or κ, and in response to immunity, the introduced human heavy and light chain transgenes have undergone class switches and somatic mutations, resulting in high affinity human IgGκ monoclonals. Producing antibodies (Lonberg, N. et al. (1994), supra; reviews in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113: 49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93 and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci. 764: 536-546). The preparation and use of HuMab mice and the genomic modifications such mice have have been described by Taylor, L. et al. (1992) Nucleic Acids, the entire contents of which are specifically incorporated herein by reference in their entirety. Research 20: 6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3720-3724; Choi et al. (1993) Nature Genetics 4: 117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152: 2912-2920; Taylor, L. et al. (1994) International Immunology 6: 579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851. Further, all US Pat. Nos. 5,545,806 to Lonberg and Kay; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; U.S. Pat. No. 5,545,807 to Surani et al .; PCT Publication WO 92/03918 to Lonberg and Kay; WO 93/12227; WO 94/25585. No., WO 97/13852, WO 98/24884 and WO 99/45962, and PCT Publication WO 01/14424 to Korman et al.

  In another embodiment, a human antibody of the invention is generated using a mouse having a human immunoglobulin sequence on the transgene and transchromosome, such as a mouse having a human heavy chain transgene and a human light chain transchromosome. can do. Such a mouse, referred to herein as “KM Mouse ™”, is described in detail in PCT Publication No. WO 02/43478 to Ishida et al.

  Still further, another transgenic animal system that expresses human immunoglobulin genes is available in the art and can be used to make the anti-CD19 antibodies of the invention. For example, another transgenic system called Xenomouse (Abgenix, Inc.) can be used; such mice are described, for example, in US Pat. Nos. 5,939,598; 6,075,181 to Kucherlapati et al., 6,114,598; 6,150,584; and 6,162,963.

  Moreover, alternative transchromosomal animal systems are available in the art and can be used to generate the anti-CD19 antibodies of the present invention. For example, a mouse having both a human heavy chain transchromosome and a human light chain transchromosome, referred to as a “TC mouse” can be used; such a mouse can be found in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97 : 722-727. In addition, bovines having human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al. (2002) Nature Biotechnology 20: 889-894) and used to produce the anti-CD19 antibodies of the present invention. Can do.

  The human monoclonal antibodies of the invention can also be prepared using phage display methods to screen libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are established in the art. For example, Ladner et al., US Pat. Nos. 5,223,409, 5,403,484, and 5,571,698; Dower et al., US Pat. Nos. 5,427,908, and 5,580,717; MaCafferty et al., US Pat. And Griffiths et al., U.S. Pat.Nos. I want to be.

  Human monoclonal antibodies of the invention can also be prepared using SCID mice so that a human antibody response can be generated upon immunization. Such mice are described, for example, in Wilson et al. US Pat. Nos. 5,476,996 and 5,698,767.

Immunization of human Ig mice When producing human antibodies of the invention using human Ig mice, Lonberg, N. et al. (1994) Nature 368 (6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14 : 845-851; and PCT publication WO 98/24884 and WO 01/14424, as described in CD19 antigen and / or recombinant CD19, or cells expressing CD19, or Such mice can be immunized by purified or concentrated preparations of the CD19 fusion protein. Preferably, the mice will be 6-16 weeks of age at the first injection. For example, human Ig mice can be immunized by intraperitoneal injection with purified or recombinant preparations (5-50 μg) of CD19 antigen.

  Detailed techniques for generating fully human monoclonal antibodies against CD19 are described in Example 1 below. The accumulation of experience with various antigens suggests that the antigen is first immunized by intraperitoneal injection with Freund's complete adjuvant, then the antigen is injected every 2 weeks with Freund's incomplete adjuvant (up to 6 total). It has been shown that transgenic mice respond. However, adjuvants other than Freund have been found to be effective as well. Furthermore, it has been found that whole cells are very immunogenic in the absence of adjuvant. The immune response can be monitored over the course of the immunization protocol by plasma samples obtained by retroorbital bleeds. Plasma can be screened by ELISA (below) and mice with sufficient titers of anti-CD19 human immunoglobulin can be used for fusion. Mice can be boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected that 2-3 fusions will need to be performed for each immunization. Typically, 6-24 mice are immunized with each antigen. Usually, both HCo7 and HCo12 lines are used. In addition, both HCo7 and HCo12 transgenes can be bred to one mouse with two different heavy chain transgenes (HCo7 / HCo12). Alternatively or additionally, the KM Mouse® strain can be used as described in Example 1.

Production of hybridomas producing human monoclonal antibodies To produce hybridomas producing the human monoclonal antibodies of the present invention, spleen cells and / or lymph node cells are isolated from immunized mice, such as mouse myeloma cell lines. It can be fused to a suitable immortal cell line. The resulting hybridomas can be screened for production of antigen-specific antibodies. For example, a single cell suspension of spleen lymphocytes from an immunized mouse is fused to 1/6 number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) by 50% PEG Can do. Alternatively, a single cell suspension of spleen lymphocytes from an immunized mouse can be obtained from a CytoPulse large chamber cell fusion electroporator (Cyto Pulse Sciences, Inc., Glen Burnie, Maryland) After seeding cells at approximately 2 x 10 ⁵ in a flat bottom microtiter plate, 20% fetal clonal serum, 18% "653" conditioned medium, 5% origin (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate In selective medium containing 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units / ml penicillin, 50 mg / ml streptomycin, 50 mg / ml gentamicin, 1 × HAT (Sigma; HAT is added 24 hours after fusion) Incubated for 2 weeks. After about 2 weeks, the cells can be cultured in a medium in which HAT is replaced with HT. Individual wells can then be screened by ELISA for human monoclonal antibodies IgM and IgG antibodies. After sufficient growth of the hybridoma has occurred, the medium can be observed usually after 10-14 days. If the hybridoma secreting the antibody is replated, screened again, and still positive for human IgG, the monoclonal antibody can be subcloned at least twice by limiting dilution. Stable subclones can then be cultured in vitro to produce small amounts of antibody in tissue culture medium for characterization.

  To purify human monoclonal antibodies, selected hybridomas can be grown in 2 L spinner flasks for monoclonal antibody purification. The supernatant can be filtered and concentrated before affinity chromatography with protein A-Sepharose (Pharmacia, Piscataway, NJ). The eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to confirm purity. The buffer can be exchanged into PBS and the concentration can be determined by OD280 using extinction coefficient 1.43. Monoclonal antibodies can be aliquoted and stored at -80 ° C.

Production of Transfectomas that Produce Monoclonal Antibodies Antibodies of the invention are also well known in the art (eg, Morrison, S. (1985) Science 229: 1202) recombinant DNA technology and gene transfection. A combination of methods can be used to produce in a host cell transfectoma.

For example, to express an antibody or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be amplified by standard molecular biology techniques (eg, using a hybridoma that expresses the antibody of interest). Or cDNA cloning) and the DNA can be inserted into an expression vector so that the gene is operably linked to transcriptional and translational control sequences. In this context, the term “operably linked” means that the antibody gene is associated with the vector such that the transcriptional and translational control sequences within the vector exhibit its intended function of regulating the transcription and translation of the antibody gene. It is intended to mean being ligated. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into different vectors or, more typically, both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (eg, ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). With light and heavy chain variable regions of the antibodies described herein, V _H segment is operatively linked to C _H segment in the vector, V _K segments functionally to the C _L segment within the vector Full-length antibody genes of any antibody isotype can be generated by inserting them into expression vectors that already encode the heavy and light chain constant regions of the desired isotype so that they bind. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

  In addition to the antibody chain gene, the recombinant expression vector of the present invention has a regulatory sequence that controls the expression of the antibody chain gene in a host cell. The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Acadamic Press, San Diego, CA (1990)). It will be appreciated by those skilled in the art that the design of an expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of host cells to be transformed, the level of expression of the desired protein, etc. . Preferred regulatory sequences for mammalian host cell expression include promoters and / or enhancers from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (eg, adenovirus major late promoter (AdMLP) and polyoma) Such viral elements that direct high level protein expression in mammalian cells are included. Alternatively, non-viral regulatory sequences such as ubiquitin promoter or β-globin promoter may be used. Still further, regulatory elements consisting of sequences from different origins such as the SRα promoter system, including sequences from the SV40 early promoter and long terminal repeats of type 1 human T cell leukemia virus (Takebe, Y. et al. (1988) Mol. Cell Biol. 8: 466-472).

  In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention have additional sequences such as sequences that regulate replication of the vector in host cells (eg, origins of replication) and selectable marker genes. Also good. The selectable marker gene facilitates selection of host cells into which the vector is introduced (see, eg, US Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells by methotrexate selection / amplification) and the neo gene (for G418 selection).

  In order to express light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. Various types of “transfection” include a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, and DEAE dextran transfection. Is intended to contain. While it is theoretically possible to express an antibody of the invention in either prokaryotic or eukaryotic host cells, expression of the antibody in eukaryotic cells and most preferably mammalian host cells may be used in such eukaryotic cells and In particular, mammalian cells are most preferred because they are more likely to assemble and secrete appropriately constructed immunologically active antibodies than prokaryotic cells. Prokaryotic expression of antibody genes has been reported to be ineffective for producing active antibodies in high yield (Boss, M.A. and Wood, C.R. (1985) Immunology Today 6: 12-13).

Preferred mammalian host cells for expressing the recombinant antibodies of the invention are described in Chinese hamster ovary (CHO cells) (eg RJ, Kaufman and PA Sharp (1982) Mol. Biol. 159: 601-621). Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, including dhfr ^- CHO cells), NSO myeloma cells, COS cells, and SP2 cells used with DHFR selectable markers Is included. In particular, another preferred expression system for use with NSO myeloma cells is WO 87/04462 (given to Wilson), WO 89/01036 (given to Bebbington), and European Patent No. This is a GS gene expression system disclosed in No. 338,841 (provided to Bebbington). When introducing a recombinant expression vector encoding an antibody gene into a mammalian host cell, a period sufficient to allow the antibody to be expressed in the host cell or, more preferably, to secrete the antibody into the medium in which the host cell is grown, Antibodies are produced by culturing host cells. Antibodies can be recovered from the culture medium using standard protein purification methods.

Characterization of Antibody Binding to Antigen Antibodies of the invention can be tested for binding to CD19, for example, by standard ELISA. Briefly, microtiter plates were coated with 0.25 μg / ml purified CD19 in PBS and then blocked with 5% bovine serum albumin in PBS. Antibody dilutions (eg, plasma dilutions from CD19 immunized mice) were added to each well and incubated at 37 ° C. for 1-2 hours. The plates were washed with PBS / Tween and then incubated for 1 hour at 37 ° C. with a secondary reagent conjugated to alkaline phosphatase (eg, for human antibodies, goat anti-human IgG Fc specific polyclonal reagent). After washing, the plates were developed with pNPP substrate (1 mg / ml) and analyzed at OD 450-650. Preferably, the mouse with the highest titer is used for fusion.

  The ELISA method described above can also be used to screen for hybridomas that show positive reaction with CD19 immunogen. A hybridoma that binds to CD19 with a high binding force is subcloned and further characterized. Select one clone from each hybridoma that retains the reactivity of the parent cells (by ELISA) to generate 5-10 vials of cells stored at -140 ° C and to purify antibodies Can do.

To purify anti-CD19 antibodies, selected hybridomas can be grown in 2 L spinner flasks for monoclonal antibody purification. The supernatant can be filtered and concentrated before affinity chromatography with Protein A-Sepharose (Pharmacia, Piscataway, NJ). The eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer can be exchanged into PBS and the concentration can be determined by OD ₂₈₀ using extinction coefficient 1.43. Monoclonal antibodies can be aliquoted and stored at -80 ° C.

  To determine the ability of selected anti-CD19 monoclonal antibodies to bind to unique epitopes, each antibody can be biotin conjugated using commercially available reagents (Pierce, Rockford, IL). As described above, competition tests using unlabeled and biotinylated monoclonal antibodies can be performed using CD19 coated ELISA plates. Biotinylated mAb binding can be detected with a streptavidin-alkaline phosphatase probe.

  To determine the isotype of the purified antibody, an isotype ELISA can be performed using reagents specific for the antibody of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, the wells of a microtiter plate were coated overnight at 4 ° C. with 1 μg / ml anti-human immunoglobulin. After blocking with 1% BSA, the plates were allowed to react for 1-2 hours at room temperature with 1 μg / ml or less of test monoclonal antibody or purified isotype control. The wells can then be reacted with human IgG or human IgM specific alkaline phosphatase binding probes. Plates are developed and analyzed as described above.

  Anti-CD19 human IgG can be further tested for reactivity against the CD19 antigen by Western blotting. Briefly, CD19 can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigen was transferred to a nitrocellulose membrane, blocked with 10% fetal calf serum, and probed with the monoclonal antibody to be tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed with BCIP / NBY substrate tablets (Sigma Chem., St. Louis, Mo).

In another aspect of the immunoconjugate , the invention features an anti-CD19 antibody or fragment thereof conjugated to a therapeutic moiety such as a cytotoxin, drug (eg, immunosuppressant) or radiotoxin. Such conjugates are referred to herein as “immunoconjugates”. Immunoconjugates that contain one or more cytotoxins are called “immunotoxins”. A cytotoxin or cytotoxic agent includes any agent that is detrimental to (eg, kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, misramycin, actinomycin D, 1 -Dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologues thereof. Therapeutic agents also include, for example, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (eg, mechloretamine, thioepachlorambucil, melphalan, Carmustine (BSNU), and Lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) (cisplatin), anthracyclines (eg, daunorubicin (this Daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, misramycin, and anthramycin (AMC)), and antimitotics (eg, Nkurisuchin and vinblastine) is included.

  Examples of other preferred therapeutic cytotoxins that can be conjugated to the antibodies of the invention include duocarmycin, calicheamicin, maytansine, and auristatin and derivatives thereof. An example of a calicheamicin antibody conjugate is commercially available (Mylotarg ™; American Home Products).

  Cytotoxins can be attached to the antibodies of the invention using linker techniques that can be used in the art. Examples of types of linkers that have been used to attach cytotoxins to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. Sensitive to cleavage by low pH within the lysosomal fraction, or against cleavage by proteases such as proteases that are selectively expressed in tumor tissues such as cathepsins (eg, cathepsins B, C, D) Sensitive linkers can be selected.

  For further discussion on cytotoxin types, methods for conjugating therapeutic agents in turn are also described by Saito, G. et al. (2003) Adv. Drug. Deliv. Rev 55: 199-215; Trail, PA et al. (2003) Cancer. Immunol. Immunother. 52: 328-337; Payne, G. (2003) Cancer Cell 3: 207-212; Allen, TM (2002) Nat. Rev. Cancer 2: 750-763; Pastan, I. and Kreitman, RJ (2002) Curr. Opin. Investig. Drugs. 3: 1089-1091; Senter, PD and Springer, CJ (2001) Adv. Drug. Deliv. Rev. 53: 247-264.

The antibodies of the present invention can also be conjugated to radioisotopes to create cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine ¹³¹ , indium ¹¹¹ , yttrium ^90, and ruthenium ¹⁷⁷ . Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates include Zevalin® (IDEC Pharmaceuticals) and Bexxar® (Corixa Pharmaceuticals), which are commercially available and using similar methods, radioactivity using the antibodies of the present invention Immune complexes can be prepared.

  The antibody conjugates of the invention can be used to modify a given biological response, and the drug moiety is not to be construed as being limited to classical therapeutic substances. For example, the drug moiety may be a protein or polypeptide having the desired biological activity. Such proteins include, for example, abrin, ricin A, an endotoxin of Pseudomonas, or an enzymatically active toxin such as diphtheria toxin or active fragments thereof; a protein such as tumor necrosis factor or interferon-γ; or For example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”) "), Granulocyte colony stimulating factor (" G-CSF "), or other growth factors.

  Techniques for attaching such therapeutic moieties to antibodies are well known, e.g., Arnon et al. `` Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy. '', `` Monoclonal Antibodies And Cancer Therapy '', Reisfeld et al. (Eds), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", "Controlled Drug Delivery (2nd Ed.)", Robinson et al. (Eds), pp 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, “Monoclonal Antibodies '84: Biological And Clinical Applications.” Pinchera et al. (Eds.), Pp. 475-506 (1985): “ Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy. '', `` Monolonal Antibodies For Cancer Detection And Therapy '', Baldwin et al. (Eds.), Pp. 303-16 (Academic Press 1985), and Thorpe Et al., Immunol. Rev., 62: 119-58 (1982).

Bispecific molecule In another aspect, the invention features a bispecific molecule comprising an anti-CD19 antibody of the invention or fragment thereof. An antibody of the invention, or antigen-binding portion thereof, is derivatized or linked to another functional molecule, such as another peptide or protein (eg, another antibody or receptor ligand), at least two different Bispecific molecules can be made that bind to a binding site or target molecule. The antibodies of the invention may actually be derivatized or linked to a plurality of other functional molecules to create multispecific molecules that bind to more than two different binding sites and / or target sites; Such multispecific molecules are also intended to be included in the term “bispecific molecule” as used herein. In order to make a bispecific molecule of the present invention, an antibody of the present invention is such as another antibody, antibody fragment, peptide, or binding mimetic, such that a bispecific molecule is obtained. It can be operably linked to one or more other binding molecules (eg, chemical coupling, genetic fusion, non-covalent association, or others).

  Accordingly, the present invention includes bispecific molecules comprising at least one first binding specificity for CD19 and a second binding specificity for a second target epitope. In certain embodiments of the invention, the second target epitope is an Fc receptor, such as human FcγRI (CD64) or human Fcα receptor (CD89). Thus, the present invention includes bispecific molecules that can bind to both FcγR or FcαR-expressing effector cells (eg, monocytes, macrophages, or polymorphonuclear cells (PMN)) and target cells that express CD19. Is included. These bispecific molecules target CD19-expressing cells to effector cells, such as phagocytosis of CD19-expressing cells, antibody-dependent cellular cytotoxicity (ADCC), cytokine release, or superoxide anion production. Elicits Fc receptor-mediated effector cell activity.

  In one embodiment of the invention in which the bispecific molecule is multispecific, the molecule can further include a third binding specificity in addition to anti-Fc binding specificity and anti-CD19 binding specificity. In one embodiment, the third binding specificity is a molecule that binds to an anti-enhancement factor (EF) moiety, eg, a surface protein involved in cytotoxic activity, thereby increasing the immune response against the target cell. An “anti-enhancement factor moiety” becomes an antibody, functional antibody fragment, or ligand that binds to a given molecule, eg, an antigen or receptor, thereby enhancing the action of a binding determinant on an Fc receptor or target cell antigen. sell. An “anti-enhancement factor moiety” can bind to an Fc receptor or a target cell antigen. Alternatively, the anti-enhancement factor moiety can bind to an entity that is different from the entity to which the first and second binding specificities bind. For example, the anti-enhancement factor moiety binds to cytotoxic T cells (eg, by CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1, or other immune cells that cause an increased immune response to the target cell) can do.

In one embodiment, the bispecific molecule of the invention comprises at least one antibody comprising as binding specificity, for example, Fab, Fab ′, F (ab ′) ₂ , Fv, Fd, dAb or single chain Fv Or an antibody fragment thereof. The antibody may also be a light or heavy chain dimer such as Fv or a single chain construct as described in US Pat. No. 4,946,778 to Ladner et al., The contents of which are specifically incorporated herein by reference. Or any minimal fragment thereof.

In one embodiment, the binding specificity for the Fcγ receptor is provided by a monoclonal antibody whose binding is not blocked by human immunoglobulin G (IgG). As used herein, the term “IgG receptor” refers to any eight gamma chain genes present on chromosome 1. These genes encode a total of 12 transmembrane or soluble receptor isoforms that fall into three Fcγ receptor classes. FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). In one preferred embodiment, the Fcγ receptor is human high affinity FcγRI. Human FcγRI is a 72 kDa molecule that exhibits high affinity (10 ⁸ to 10 ⁹ M ⁻¹ ) for monomeric IgG.

  The production and characterization of certain preferred anti-Fcγ monoclonal antibodies is described in PCT publication WO 88/00052 and US Pat. No. 4,954,617 to Fanger et al., The teachings of which are hereby incorporated by reference in their entirety. Described. These antibodies bind to an epitope of FcγRI, FcγRII, or FcγRIII at a site different from the receptor's Fcγ binding site, and thus the binding is not substantially blocked by physiological levels of IgG. Specific anti-FcγRI antibodies useful in the present invention are mAb 22, mAb 32, mAb 44, mAb 62, and mAb 197. A hybridoma producing mAb 32 is available from American Type Culture Collection ATCC Accession No. HB 9469. In one embodiment, the anti-Fcγ receptor antibody is a humanized form of monoclonal antibody 22 (H22). Production and characterization of the H22 antibody is described in Graziano, R.F. et al. (1995) J. Immunol. 155 (10): 4996-5002 and PCT publication WO 94/10332 granted to Tempest et al. The H22 antibody-producing cell line has been deposited with the American Type Culture Collection under the name HA022CL1 and has the accession number CRL 11177.

In yet another preferred embodiment, the binding specificity of the Fc receptor binds to a human IgA receptor, such as an Fc-α receptor (FcαRI (CD89)) whose binding is preferably not blocked by human immunoglobulin A (IgA). Provided by the antibody. The term “IgA receptor” is intended to include the gene product of one α gene (FcαRI) present on chromosome 19. This gene is known to encode several alternatively spliced transmembrane isoforms of 55-110 kDa. FcαRI (CD89) is constitutively expressed on monocytes / macrophages, eosinophils and neutrophil granulocytes, but not on non-effector cell populations. FcαRI has a moderate affinity (˜5 × 10 ⁷ M ⁻¹ ) for both IgA1 and IgA2, which increases after exposure to cytokines such as G-CSF or GM-CSF (Morton, HC (1996) Critical Reviews in Immunology 16: 423-440). Four FcαRI-specific monoclonal antibodies identified as A3, A59, A62, and A77 that bind to FcαRI outside the IgA ligand binding domain have been described (Monteiro, RC et al. (1992) J. Immunol. 148: 1764 ).

  FcαRI and FcγRI are (1) expressed predominantly in immune effector cells such as monocytes, PMNs, macrophages, and dendritic cells, (2) expressed at high levels (eg, 5,000 to 100,000 per cell) Are mediators of cytotoxic activity (eg, ADCC, phagocytosis), and (4) mediate enhancement of antigen presentation of antigens, including self-antigens targeted to them. A preferred triggering receptor for use in bispecific molecules.

  Although human monoclonal antibodies are preferred, other antibodies that can be used in the bispecific molecules of the invention are mouse, chimeric, and humanized monoclonal antibodies.

  Bispecific molecules of the present invention can be prepared by binding component binding specificities, such as anti-FcR and anti-CD19 binding specificities, using methods known in the art. For example, each binding specificity of a bispecific molecule can be produced individually and bound to each other. If the binding specificity is a protein or peptide, various coupling agents or cross-linking agents can be used for covalent binding. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), o-phenylene dimaleimide (oPDM) ), N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), and sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) (eg Karpovsky et al. ( 1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82: 8648). Other methods include Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229: 81-83 and Glennie et al. (1987) J. Immnol. 139: 2367-2375. The methods described are included. Preferred binding materials are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

  If the binding specificity is an antibody, they can be linked by sulfhydryl binding of the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to include an odd, preferably one sulfhydryl residue, prior to conjugation.

Alternatively, both binding specificities are encoded in the same vector and expressed and constructed in the same host cell. This method is particularly useful when the bispecific molecule is a mAb × mAb, mAb × Fab, Fab × F (ab ′) ₂ , or ligand × Fab fusion protein. The bispecific molecule of the present invention can be a single chain molecule comprising one single chain antibody and a binding determinant, or can be a single chain bispecific molecule comprising two binding determinants. Bispecific molecules include at least two single chain molecules. Methods for preparing bispecific molecules include, for example, U.S. Patent No. 5,260,203, U.S. Patent No. 5,455,030, U.S. Patent No. 4,881,175, U.S. Patent No. 5,132,405, U.S. Patent No. 5,091,513, U.S. Patent No. 5,476,786, U.S. Pat. No. 5,013,653, US Pat. No. 5,258,498, and US Pat. No. 5,482,858, all of which are hereby incorporated by reference in their entirety.

  Binding of the bispecific molecule to its specific target can be achieved by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (eg, growth inhibition) or Western blot assay. Can be confirmed. Each of these assays generally detects the presence of a protein-antibody complex of interest, in particular, by using a labeling reagent (eg, an antibody) specific for the complex of interest. For example, the FcR-antibody complex can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the antibody-FcR complex. Alternatively, the complex can be detected using any of a variety of other immunoassay methods. For example, antibodies can be radioactively labeled and used in radioimmunoassay (RIA) (eg, Weinstraub, B. “Principles of Radioimmunoassays”, Seventh Training Course on Radioligand Assay Techniques, incorporated herein by reference. See The Endocrine Society, March 1986). The radioactive isotope can be detected by such means as the use of a γ counter or a scintillation counter or autoradiography.

In another aspect, the invention provides a composition comprising one or a combination of the monoclonal antibodies of the invention, or antigen-binding portions thereof, formulated with a pharmaceutically acceptable carrier, for example, A pharmaceutical composition is provided. Such compositions may include one or a combination (eg, two or more different) antibodies, immunoconjugates, or bispecific molecules of the invention. For example, a pharmaceutical composition of the invention can comprise a combination of antibodies (or immunoconjugates or bispecific molecules) that bind to different epitopes on the target antigen or that have complementary activities.

  The pharmaceutical compositions of the present invention can also be administered in combination therapy, ie in combination with other substances. For example, combination therapy can include an anti-CD19 antibody of the invention in combination with at least one other anti-inflammatory or immunosuppressive agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail in the sections below on the use of the antibodies of the invention.

  As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents that are physiologically compatible, As well as absorption delaying agents and the like. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or intraepidermal administration (eg, injection or infusion). Depending on the route of administration, the active compound, i.e. antibody, immune complex, or bispecific molecule, is coated with a material that protects the compound from the action of acids and other natural conditions that may inactivate the compound. May be.

  The pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable salts. “Pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart undesired toxic effects (see, eg, Berge, SM et al. (1977) J. Pharm. Sci. 66 : 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, aromatics, as well as non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorus. Salts derived from non-toxic organic acids such as aliphatic acids, aliphatic and aromatic sulfonic acids and the like are included. Base addition salts include N, N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, chlorine, diethanolamine, ethylenediamine, and procaine along with alkaline earth metals such as sodium, potassium, magnesium, and calcium And salts derived from non-toxic organic amines such as

  The pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like, (2) ascorbyl palmi Fat-soluble antioxidants such as tate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol, and (3) citric acid, ethylenediaminetetraacetic acid (EDTA) , Metal chelators such as sorbitol, tartaric acid, phosphoric acid and the like.

  Examples of suitable water-soluble and water-insoluble carriers that may be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), and suitable mixtures thereof, olive oil Vegetable oils such as, and injectable organic esters such as ethyl oleate. For example, proper fluidity can be maintained by using a coating material such as lecithin, by maintaining the required particle size in the case of a dispersant, and by using a surfactant.

  These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured by the sterilization techniques described above and by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, and phenol sorbic acid. Similarly, it may be desirable to add isotonic agents such as sugar and sodium chloride to the composition. In addition, sustained absorption forms of injectable preparations may be obtained by including substances which delay absorption such as aluminum monostearate and gelatin.

  Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and materials for pharmaceutically active substances is known in the art. Except insofar as any conventional media or substance is incompatible with the active compound, its use in the pharmaceutical compositions of the present invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

  The therapeutic composition typically must be sterile and stable under the conditions of manufacture and storage. The composition can be prepared as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

  Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent with one or a combination of the ingredients listed above and then microfiltering as necessary. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for preparing sterile injectable solutions, the preferred method of preparation is vacuum drying and lyophilization (lyophilization), which yields a powder of the active ingredient plus any further desired ingredients from the already filter sterilized solution. .

  The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Generally in 100%, this amount is from about 0.01% to about 99% of the active ingredient, preferably from about 0.1 to about 70%, most preferably about 1% of the active ingredient in combination with a pharmaceutically acceptable carrier. It appears to be in the range of 1% to about 30%.

  Dosage regimens are adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be reduced or increased proportionally depending on the urgency of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Unit dosage forms as used herein refer to physically discrete units suitable as unit dosages for the subject to be treated; each unit has the desired therapeutic effect in relation to the required pharmaceutical carrier. Contains a predetermined amount of active ingredient calculated to occur. The specifications for the unit dosage forms of the present invention include: (a) the unique characteristics of the active compound and the specific therapeutic effect obtained, and (b) when synthesizing such an active compound for the treatment of sensitivity in an individual. It depends on and is directly dependent on limitations inherent in the art.

  When administering the antibody, the dosage ranges from about 0.0001 to 100 mg / kg, more usually 0.01 to 5 mg / kg host body weight. For example, the dose can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, or 10 mg / kg body weight, or can be in the range of 1-10 mg / kg. Exemplary treatment regimens are once a week, once every two weeks, once every three weeks, once every four weeks, once every month, once every three months, or every 3-6 months Includes one dose. A preferred dosing regimen for anti-CD19 antibodies of the invention includes administering 1 mg / kg body weight or 3 mg / kg body weight by intravenous administration, and the antibody is administered using one of the following dosing schedules: (I) administered 6 times every 4 weeks, then every 3 months; (ii) every 3 weeks; (iii) 3 mg / kg body weight followed by 1 mg / kg body weight every 3 weeks.

  In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered falls within the indicated range. The antibody is usually administered multiple times. The dosing interval can be, for example, weekly, monthly, every three months or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibodies against the target antigen in the patient. In some methods, dosage is adjusted to obtain an antibody plasma concentration of about 1-1000 μg / ml and in some methods about 25-300 μg / ml.

  Alternatively, the antibody can be administered as a sustained release formulation, in which case less frequent administration is required. The dose and frequency of administration will vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. For prophylactic applications, a relatively low dose is administered over a relatively long period of time. Some patients continue to receive treatment for the rest of their lives. For therapeutic applications, it is sometimes necessary to administer relatively high doses in a relatively short period of time until disease progression is reduced or stopped, and preferably until the patient shows partial or complete improvement of disease symptoms It is. Thereafter, the patient can be administered a prophylactic regime.

  The actual dosage level of the active ingredient in the pharmaceutical composition of the invention is effective to obtain the desired therapeutic response, composition, and mode of administration for a particular patient without toxic to the patient. It may vary to obtain component amounts. The selected dose level depends on the particular composition of the invention used, or the activity of the ester, salt, or amide, route of administration, administration time, excretion rate of the particular compound used, duration of treatment, other drugs used Includes compounds and / or materials used in conjunction with a particular composition, the age, sex, weight, condition, general health and history of the patient being treated, and similar factors well known in the medical arts It is thought that it depends on various pharmacokinetic factors.

A “therapeutically effective amount” of an anti-CD19 antibody of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the number and duration of disease-free periods, or prevention of disorders or incompetence due to the disease. For example, for the treatment of CD19 ⁺ tumors, a “therapeutically effective amount” preferably has a cell growth or tumor growth of at least about 20%, more preferably at least about 40%, even more preferably compared to an untreated subject. Inhibits at least about 60%, and even more preferably at least about 80%. Whether a compound can inhibit tumor growth can be assessed in animal model systems that predict efficacy in human tumors. Alternatively, this property of the composition can be assessed by examining the ability of the compound to inhibit cell growth, and such inhibition can be measured in vitro by assays known to those skilled in the art. A therapeutically effective amount of the therapeutic compound can reduce the size of the tumor in the subject or otherwise ameliorate the symptoms. One of skill in the art would be able to determine such amounts based on factors such as the subject's physique, the severity of the subject's symptoms, and the particular composition or route of administration selected.

  The compositions of the present invention can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route of administration and mode of administration will vary depending on the desired result. Preferred routes of administration of the antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, for example by injection or infusion. As used herein, the phrase “parenteral administration” usually refers to modes of administration other than enteral and topical administration by injection, including intravenous, intramuscular, intraarterial, intrathecal. These include intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, epidermal, intraarticular, intracapsular, subarachnoid, intrathecal, epidural, and intracisternal injection and infusion It is not limited to.

  Alternatively, the antibodies of the invention may be administered by a parenteral route, such as a topical, epidermal, or intramucosal route of administration, such as intranasal, oral, intravaginal, rectal, sublingual, or topical. Can do.

  The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for the preparation of such formulations have been patented or are generally known to those skilled in the art. See, for example, “Sustained and Controlled Release Drug Delivery Systems”, J.R. Robinson, ed. Marcel Dekker, Inc., New York, 1978.

  The therapeutic composition can be administered by medical devices known in the art. For example, in a preferred embodiment, the therapeutic composition of the present invention comprises U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Can be administered by a hypodermic injection device without a needle, such as the device disclosed in US Pat. Examples of well known implants and modules useful in the present invention include the following: US Pat. No. 4,487,603 disclosing an implantable microinfusion pump for dispensing a drug at a controlled rate, administering a drug through the skin U.S. Pat. No. 4,486,194 disclosing a therapeutic device for performing, U.S. Pat. No. 4,447,233 disclosing a drug infusion pump for delivering a drug at an accurate infusion rate, variable flow rate implantable infusion device for sustained drug delivery U.S. Pat. No. 4,447,224, U.S. Pat. No. 4,439,196 which discloses an osmotic drug delivery system having a multi-compartment compartment, and U.S. Pat. No. 4,475,196 which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

  In certain embodiments, human monoclonal antibodies of the invention can be prepared to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many very hydrophilic compounds. In order to ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be prepared, for example, in liposomes. See, for example, US Pat. Nos. 4,522,811, 5,374,548, and 5,399,331 for methods of producing liposomes. Liposomes may contain one or more moieties that are selectively transported to specific cells or organs and thus enhance targeted drug delivery (eg, VV Ranade (1989) J. Clin. Pharmacol. 29 : 685). Exemplary targeting moieties include folate or biotin (see, eg, US Pat. No. 5,416,016 to Low et al.), Mannoside (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153: 1038), antibodies (PG Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180), surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134), p120 (Schreier et al. (1994) J. Biol. Chem. 269: 9090); K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346: 123; JJ Killion, IJ Fidler ( (1994) Immunomethods 4: 273.

Applications and Methods The antibodies, particularly human antibodies, antibody compositions, and methods of the present invention have numerous in vitro and in vivo diagnostic and therapeutic utilities involving the diagnosis and treatment of CD19 mediated disorders. For example, these molecules can be administered to cultured cells in vitro or ex vivo, or for example, in vivo to human subjects to treat, prevent, and diagnose a variety of disorders. As used herein, the term “subject” is intended to include human and non-human animals. Non-human animals include all vertebrates, eg, mammals and non-mammals such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. Preferred subjects include human patients with disorders mediated by CD19 activity. The method is particularly suitable for treating human patients with disorders associated with abnormal CD19 expression. When an antibody against CD19 is administered with another substance, both can be administered in either order or simultaneously.

  Given the specific binding of the antibodies of the invention with respect to CD19, the antibodies of the invention can be used to specifically detect CD19 expression on the cell surface, as well as to purify CD19 by immunoaffinity purification. Can be used.

  Furthermore, considering the expression of CD19 in various tumor cells, the human antibodies, antibody compositions, and methods of the present invention can be used for tumorigenic disorders such as non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL). ), Chronic lymphocytic leukemia (CLL), Burkitt lymphoma, undifferentiated large cell lymphoma (ALCL), multiple myeloma, cutaneous T-cell lymphoma, nodular small cut nucleated cell lymphoma, lymphocytic lymphoma, Peripheral T-cell lymphoma, Renato lymphoma, immunoblastic lymphoma, T-cell leukemia / lymphoma (ATLL), adult T-cell leukemia (T-ALL), entroblastic / centrocytic (centrocytic) cb / cc) follicular lymphoma cancer, diffuse large B-cell lymphoma, angioimmunoblastic lymphadenopathy (AILD) -like T-cell lymphoma, HIV-related coelomic lymphoma, fetal cancer, nasopharyngeal fraction Characterized by the presence of tumor cells that express CD19, including metaplastic cancers (eg Schminke tumors), Castorman disease, Kaposi's sarcoma, multiple myeloma, Waldenstrom globulinemia and other B-cell lymphomas Can be used to treat subjects with disabilities.

Furthermore, overexpression of CD19 is a loss of B cell tolerance may generate autoimmune disorders (Tedder et al (2005) Curr Dir Autoimmun 8:. 55). This autoimmune effect was confirmed by the accumulation of CD19 + B cells in inflamed joints of patients with rheumatoid arthritis (He et al. (2001) J Rheumatol 28 : 2168). Accordingly, the human antibodies, antibody compositions and methods of the invention can be used to treat a subject having an autoimmune disorder, eg, a disorder characterized by the presence of B cells that express CD19, eg, rheumatoid arthritis.

  In one embodiment, the antibodies of the invention (eg, human monoclonal antibodies, multispecific and bispecific molecules and compositions) are used to detect CD19 levels or cell levels that contain CD19 on their membrane surface. Can then be linked to that particular disease symptom. Alternatively, antibodies can be used to inhibit or block CD19 function, which can then be linked to the prevention or amelioration of specific disease symptoms, thereby implicating CD19 as a disease mediator. This can be done by contacting the sample and control sample with an anti-CD19 antibody under conditions that allow the formation of a complex of antibody and CD19. Any complexes formed between the antibody and CD19 are detected and compared in the sample and control.

  In another embodiment, antibodies of the invention (eg, human antibodies, multispecific and bispecific molecules and compositions) are first tested for binding activity associated with in vitro therapeutic or diagnostic uses. Can do. For example, the compositions of the invention can be tested using the flow cytometry assay described in the examples below.

  The antibodies (eg, human antibodies, multispecific and bispecific molecules, immunoconjugates, and compositions) of the present invention have additional utility in the treatment and diagnosis of CD19 related diseases. For example, human monoclonal antibodies, multispecific or bispecific molecules and immunoconjugates can be used to elicit one or more of the following biological activities in vivo or in vitro: for cells expressing CD19 Activity to inhibit proliferation and / or kill cells expressing CD19; activity to mediate phagocytosis or ADCC of cells expressing CD19 or block CD19 ligand binding to CD19 in the presence of human effector cells.

  In certain embodiments, antibodies (eg, human antibodies, multispecific and bispecific molecules and compositions) are used in vivo to treat, prevent, or diagnose a variety of CD19 related diseases. Examples of CD19-related diseases include autoimmune diseases, joint immunity, cancer, non-Hodgkin lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), Burkitt lymphoma, undifferentiated large cell lymphoma (ALCL), multiple myeloma, cutaneous T-cell lymphoma, nodular small cell nuclear lymphoma, lymphocytic lymphoma, peripheral T-cell lymphoma, Renato lymphoma, immunoblastic lymphoma, T-cell leukemia / Lymphoma (ATLL), adult T-cell leukemia (T-ALL), entropic / centrosite (cb / cc) follicular lymphoma cancer, diffuse large B-cell lymphoma, angioimmunoblastic lymphadenopathy (AILD) -Like T-cell lymphoma, HIV-related body cavity lymphoma, fetal cancer, nasopharyngeal undifferentiated cancer (eg, Schminke tumor), Castorman's disease, Kaposi's sarcoma, multiple myeloma, Waldenstrom's large globulin Hyperlipidemia, and other B- cell lymphomas.

  Suitable routes for administering the antibody compositions of the invention (eg, human monoclonal antibodies, multispecific and bispecific molecules and immune complexes) in vivo and in vitro are well known in the art, Can be selected by a vendor. For example, the antibody composition can be administered by injection (eg, intravenous or subcutaneous). The appropriate dose of the molecule used will depend on the age and weight of the subject and the concentration and / or formulation of the antibody composition.

  As described above, the human anti-CD19 antibodies of the present invention can be co-administered with one or more other therapeutic agents, such as cytotoxic agents, radioactive agents, or immunosuppressive agents. The antibody can be linked to the substance (as an immune complex) or can be administered separately from the substance. In the latter case (separate administration), the antibody can be administered before, after, or simultaneously with the substance, or can be co-administered with other known treatments, such as anti-cancer treatments, such as radiation therapy. Such therapeutic agents include, among others, doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil, and cyclophosphamide, hydroxyurea, which are effective only to levels that are toxic or subtoxic to the patient. Antineoplastic agents such as Cisplatin is intravenously administered as a 100 mg / dose once every 4 weeks, and adriamycin is intravenously administered as a 60-75 mg / ml dose every 21 days. Co-administration of the human anti-CD19 antibody or antigen-binding fragment thereof of the present invention and a chemotherapeutic agent provides two anticancer agents that are manipulated by different mechanisms that produce cytotoxic effects on human tumor cells. Such co-administration can solve problems due to the development of resistance to drugs or changes in the antigenicity of tumor cells that would render them non-responsive to antibodies.

Target-specific effector cells, such as effector cells linked to compositions of the invention (eg, human antibodies, multispecific and bispecific molecules) can also be used as therapeutic agents. Effector cells for targeting can be human leukocytes such as macrophages, neutrophils, or monocytes. Other cells include eosinophils, natural killer cells, and cells with other IgG or IgA receptors. If desired, effector cells can be obtained from the subject to be treated. Target-specific effector cells can be administered as a cell suspension in a physiologically acceptable solution. The number of cells administered can be on the order of 10 ⁸ to 10 ^9, but this will vary depending on the therapeutic purpose. In general, the amount will be sufficient to obtain localization in target cells, eg, tumor cells expressing CD19, and to perform cell killing, eg, by phagocytosis. The route of administration can vary as well.

  Treatment with target-specific effector cells can be performed in conjunction with other techniques for removing target cells. For example, anti-tumor treatments using the compositions of the invention (eg, human antibodies, multispecific and bispecific molecules) and / or effector cells having these compositions can be used in conjunction with chemotherapy. In addition, combination immunotherapy may be used to direct two different cytotoxic effector populations to tumor cell rejection. For example, anti-CD19 antibodies linked to anti-FcγRI or anti-CD3 may be used with IgG or IgA receptor specific binding substances.

  Bispecific and multispecific molecules of the present invention can also be used to modulate FcγR or FcγR levels on effector cells, such as by receptor capping and loss on the cell surface. Mixtures of anti-Fc receptors can be used for this purpose as well.

  Compositions of the invention having complement binding sites such as IgG1, -2, or -3, or portions from IgM that bind complement (eg, human antibodies, multispecific and bispecific molecules and The immune complex) can also be used in the presence of complement. In one embodiment, ex vivo treatment of a cell population comprising target cells with a binding agent of the invention and appropriate effector cells can be supplemented by adding complement or serum containing complement. Phagocytosis of target cells coated with a binding agent of the invention can be improved by binding of complement proteins. In another embodiment, target cells coated with the compositions of the invention (eg, human antibodies, multispecific and bispecific molecules) can also be lysed by complement. In yet another embodiment, the compositions of the invention do not activate complement.

  The compositions of the invention (eg, human antibodies, humanized antibodies, or chimeric antibodies, multispecific and bispecific molecules and immune complexes) can also be administered with complement. In certain embodiments, the present disclosure provides a composition comprising a human antibody, a multispecific or bispecific molecule, and serum or complement. These compositions may be advantageous when the complement is present very proximal to the human antibody, multispecific or bispecific molecule. Alternatively, the human antibodies, multispecific or bispecific molecules of the invention and complement or serum can be administered separately.

  Kits comprising an antibody composition of the invention (eg, a human antibody, bispecific or multispecific molecule, or immunoconjugate) and instructions for use are likewise within the scope of the invention. The kit further comprises an immunosuppressant, cytotoxic agent, or radioactive agent, or one or more additional human antibodies of the invention (eg, complement activity that binds to a different epitope on the CD19 antigen than the first human antibody). One or more additional reagents such as

  Accordingly, the patient treated with the antibody composition of the present invention is further administered with another therapeutic substance such as a cytotoxic substance or radioactive disorder that enhances or enhances the therapeutic effect of the human antibody (of the human antibody of the present invention). Before, simultaneously, or after).

  In other embodiments, the subject can be further treated with an agent that modulates, eg, enhances or inhibits, the expression or activity of Fcγ or Fcγ receptor, eg, by treating the subject with a cytokine. Preferred cytokines to administer during treatment with multispecific molecules include granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-γ (IFN-γ) , And tumor necrosis factor (TNF).

  The compositions of the invention (eg, human antibodies, multispecific and bispecific molecules) can also be used to target cells that express FcγR or CD19, eg, to label such cells. it can. For such applications, the binding agent can be linked to a molecule that can be detected. Thus, the present invention provides methods for localizing ex vivo or in vitro cells that express FcγR, or Fc receptors such as CD19. The detectable label can be, for example, a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.

  In certain embodiments, the invention contacts a sample and a control sample with a human monoclonal antibody that specifically binds CD19, or an antigen-binding fragment thereof, under conditions that allow the antibody or a portion thereof to form a complex with CD19. Providing a method for detecting the presence of CD19 antigen in a sample or for determining the amount of CD19 antigen. Next, complex formation is detected and if the complex formation differs between samples compared to a control sample, it indicates that CD19 antigen is present in the sample.

  In other embodiments, the present invention provides a CD19-mediated disorder in a subject, such as autoimmune disease, rheumatoid arthritis, cancer, non-Hodgkin's lymphoma, acute lymphocytic leukemia ( ALL), chronic lymphocytic leukemia (CLL), Burkitt lymphoma, undifferentiated large cell lymphoma (ALCL), cutaneous T-cell lymphoma, nodular small cut nucleated cell lymphoma, lymphocytic lymphoma, peripheral T- Cellular lymphoma, Renato lymphoma, Immunoblastic lymphoma, T-cell leukemia / lymphoma (ATLL), Adult T-cell leukemia (T-ALL), Entroptic / centrosite (cb / cc) follicular lymphoma cancer, Diffuse Large cell B-cell lymphoma, angioimmunoblastic lymphadenopathy (AILD) -like T-cell lymphoma, HIV-related body cavity lymphoma, fetal cancer, nasopharyngeal anaplastic cancer (eg, Schminke瘍), provides Castleman's disease, Kaposi's Sarcoma, multiple myeloma, a method for treating Waldenstrom macroglobulinemia and other B- cell lymphomas. Such antibodies and derivatives thereof are used to inhibit CD19-induced activity associated with certain disorders, such as proliferation and differentiation. By contacting the antibody with CD19 (eg, by administering the antibody to a subject), the ability of CD19 to induce such activity is inhibited, thus treating the associated disorder. The antibody composition can be administered alone, or with another therapeutic agent, such as a cytotoxic or radioactive disorder that acts synergistically with the antibody composition to treat or prevent CD19 mediated diseases. Can be administered.

  In yet another embodiment, the immunoconjugate of the invention has a CD19 cell surface receptor by linking a compound (eg, therapeutic agent, label, cytotoxin, radiotoxin, immunosuppressive agent, etc.) to the antibody. It can be used to target such compounds to cells. For example, an anti-CD19 antibody can be prepared by using U.S. Patent No. 6,281,354, U.S. Patent No. 6,548,530, U.S. Patent Application Publication No. 20030050331, U.S. Patent Application Publication No. 20030064984, U.S. Patent Application Publication No. 20030073852, U.S. Patent Application Publication No. 20040087497. Or can be bound to any of the toxin compounds described in WO 03/022806. Thus, the present invention also provides for localizing CD19-expressing cells ex vivo or in vivo (eg, by a detectable label such as a radioisotope, fluorescent compound, enzyme, or enzyme cofactor). Provide a method. Alternatively, immunoconjugates can be used to kill cells with CD19 cell surface receptors by targeting cytotoxins or radiotoxins to CD19.

  The invention is further illustrated by the following examples, which should not be construed in a more restrictive manner. The contents of all drawings and all references, patents, and published patent applications cited throughout this application are specifically incorporated herein by reference.

Example
Example 1 : Production of human monoclonal antibody against CD19
B cell tumor cells Raji (ATCC accession number CCL-86) and Daudi (ATCC accession number CCL-213) were used as antigens for antigen immunization.

Transgenic transgenic chromosomal KM-MOUSE (registered trademark)
A fully human monoclonal antibody against CD19 was prepared using a transgenic chromosomal mouse of the KM strain that expresses the human antibody gene. This mouse strain was homozygously disrupted by the endogenous mouse kappa light chain gene as described in Chen et al. (1993) EMBO J. 12: 811-820, and the international publication of the PCT publication on HuMab mice. As described in Example 1 of publication 01/09187, the endogenous mouse heavy chain gene is homozygously disrupted. This mouse carries the human kappa light chain transgene KCo5 as described in Fishwild et al. (1996) Nature Biotechnology 14: 845-851. This mouse also carries the human heavy chain transchromosome SC20 as described in PCT publication WO 02/43478.

KM-MOUSE® immunity:
To generate fully human monoclonal antibodies against CD19, the KM-MOUSE® population was immunized with either Raji or Daudi B cell tumor cell lines. General immunization schemes are described in Lonberg, N. et al. (1994) Nature 368 (6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14 : 845-851 and the PCT publication. It is described in publication 98/24884. Mice were 6-16 weeks of age at the first antigen injection. Mice (KM-MOUSE®) were immunized intraperitoneally (IP) with the cell preparation.

  Transgenic mice are immunized twice with antigen in complete Freund's adjuvant or Ribi adjuvant, followed by IP immunization for 3-21 days with antigen in incomplete floyd adjuvant or Ribi adjuvant (up to a total of 11 immunizations) Went. The immune response was observed by retroorbital bleeds. Plasma was screened by ELISA (as described below) and mice with sufficient anti-CD19 human immunoglobulin titers were used for fusion. Mice were boosted intravenously with antigen 3 days before sacrifice and removal of the spleen.

Selection of anti-CD19 antibody producing KM-MOUSE® 0:
Serum from immunized mice by a modified ELISA first described by Fishwild, D. et al. (1996) (supra) to screen for KM-MOUSE® producing antibodies that bind to CD19 Was tested. Briefly, microtiter plates were coated with 1-2 μg / ml purified recombinant CD19 fusion protein in PBS, 50 μl / well incubated at 4 ° C. overnight, then blocked with 200 μl / well PBS containing 5% BSA. . Plasma dilutions from CD19 immunized mice were added to each well and incubated for 1-2 hours at ambient temperature. The plates were washed with PBS / Tween and then incubated with alkaline phosphatase-conjugated goat anti-human kappa light chain polyclonal antibody for 1 hour at room temperature. After washing, the plates were developed with pNPP substrate and analyzed at OD 415-650 using a spectrophotometer. Mice that showed the highest anti-CD19 antibody titer were used for fusion. Fusion was performed as described below, and hybridoma supernatants were tested for anti-CD19 activity using ELISA.

Production of a hybridoma producing a human monoclonal antibody against CD19:
Mouse splenocytes isolated from KM-MOUSE® can be obtained using PEG based on standard protocols or Cyto Pulse large chamber cell fusion electroporator (Cyto Pulse Sciences , Inc., Glen Burnie, MD) and fused to a mouse myeloma cell line with PEG using electric field electrofusion. The resulting hybridomas were screened for production of antigen specific antibodies. Single cell suspensions of splenic lymphocytes from immunized mice were fused to a quarter number of SP2 / 0 nonsecreting mouse myeloma cells (ATCC, CRL 1581) with 50% PEG (Sigma). Cells were seeded at approximately 1 × 10 ⁵ cells / well in a flat bottom microtiter plate and DMEM (Mediatech, CRL 10013, high glucose, L-glutamine, and sodium pyruvate) in 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 10% fetal bovine serum, 10% P388D1 (ATCC, CRL TIB-63) conditioned medium, 3-5% origen (IGEN) in 50 mg / ml gentamicin and 1 × HAT (Sigma, CRL P-7185) ) For about 2 weeks. After 1-2 weeks, the cells were cultured in a medium in which HAT was replaced with HT. Individual wells were screened by ELISA (above) for human anti-CD19 monoclonal IgG antibody. After sufficient hybridoma growth occurred, the medium was monitored usually after 10-14 days. Hybridomas secreting the antibody were replated and screened again, and if still positive for human IgG, the anti-CD19 monoclonal antibody was subcloned at least twice by limiting dilution. Stable subclones were cultured in vitro to produce small amounts of antibody in tissue culture medium for further characterization.

  Hybridoma clones 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8 made from HuMAb mice® were selected for further analysis.

Example 2 : Structural characterization of human monoclonal antibodies 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8
CDNA sequences encoding the heavy and light chain variable regions of 21D4 and 21D4a monoclonal antibodies are obtained from 21D4 hybridomas using standard PCR techniques and sequenced using standard DNA sequencing techniques. It was. Note that the 21D4 hybridoma produces an antibody having a heavy chain paired with one of two light chains (SEQ ID NOs: 8 and 9). Both antibodies (i.e., SEQ ID NO: 21D4 having V _H and V _L sequences each of 1 and 8, and SEQ ID NO: 21D4a having V _H and V _L sequences each of 1 and 9) in the CD19 Join. The cDNA sequences encoding the heavy and light chain variable regions of the 47G4, 27F3, 3C10, 5G7, 13F1 and 46E8 monoclonal antibodies are respectively 21D4, 21D4a, 47G4, 27F3, 3C10, 5G7 using standard PCR techniques. , Obtained from 13F1 and 46E8 hybridomas and sequenced using standard DNA sequencing techniques.

  The nucleotide and amino acid sequences of the heavy chain variable region of 21D4 are shown in FIG. 1A and are shown in SEQ ID NOs: 59 and 1, respectively.

  The nucleotide and amino acid sequences of the light chain variable region of 21D4 are shown in FIG. 1B and are shown in SEQ ID NOs: 66 and 8, respectively.

Comparison of 21D4 heavy chain immunoglobulin sequences with known human germline immunoglobulin heavy chain sequences shows that the 21D4 heavy chain is derived from the V _H segment from human germline V _H 5-51, from human germline 3-10 We have demonstrated the use of the D segment and the J _H segment from human germline JH4b. The alignment of the 21D4 V _H sequence with the germline V _H 5-51 sequence is shown in FIG. Further analysis of the 21D4 V _H sequence using the Kabat CDR region determination is shown in FIGS. 1A and 8 and the CDR1 region, CDR2 region and CD3 region of the heavy chain as shown in SEQ ID NOs: 16, 23 and 30 respectively. It led to depiction.

Comparison of the 21D4 light chain immunoglobulin sequence with the known human germline immunoglobulin light chain sequence shows that the 21D4 light chain contains the _VL segment from human germline V _K L18 and the J _K segment from human germline JK2. It has been demonstrated to be used. The alignment of the 21D4 V _L sequence and the germline V _K L18 sequence is shown in FIG. Further analysis of the 21D4 _VL sequence using Kabat CDR region determination is shown in FIGS. 1B and 15 and in the light chain CDR1, CDR2 and CD3 regions as shown in SEQ ID NOs: 37, 44 and 51, respectively. It led to depiction.

  The nucleotide and amino acid sequences of the heavy chain variable region of 21D4a are shown in FIG. 1A and are shown in SEQ ID NOs: 59 and 1, respectively.

  The nucleotide and amino acid sequences of the light chain variable region of 21D4a are shown in FIG. 1C and are shown in SEQ ID NOs: 67 and 9, respectively.

Comparison of the 21D4a heavy chain immunoglobulin sequence with the known human germline immunoglobulin heavy chain sequence shows that the 21D4a heavy chain is derived from the human germline V _H 5-51 V _H segment, from human germline 3-10 We have demonstrated the use of the D segment and the J _H segment from human germline JH4b. The alignment of the 21D4a V _H sequence with the germline V _H 5-51 sequence is shown in FIG. Further analysis of the 21D4a V _H sequence using the Kabat CDR region determination shows that the CDR1 region, CDR2 region and CD3 region of the heavy chain as shown in FIGS. It led to depiction.

Comparison of 21D4a light chain immunoglobulin sequence to the known human germline immunoglobulin light chain sequences, the J _K segment from V _L segments and the human germline JK3 from 21D4a light chain human germline V _K L18 It has been demonstrated to be used. The alignment of the 21D4a V _L sequence and the germline V _K L18 sequence is shown in FIG. Further analysis of the 21D4a V _L sequence using the Kabat CDR region determination shows that the CDR1, CDR2 and CD3 regions of the light chain as shown in FIGS. 1C and 16 and SEQ ID NOs: 37, 44 and 52, respectively. It led to depiction.

  The nucleotide and amino acid sequences of the heavy chain variable region of 47G4 are shown in FIG. 2A and are shown in SEQ ID NOs: 60 and 2, respectively.

  The nucleotide and amino acid sequences of the light chain variable region of 47G4 are shown in FIG. 2B and are shown in SEQ ID NOs: 68 and 10, respectively.

A comparison of the 47G4 heavy chain immunoglobulin sequence with the known human germline immunoglobulin heavy chain sequence shows that the 47G4 heavy chain is derived from the human germline V _H 1-69 V _H segment, from the human germline 6-19 We have demonstrated the use of the D segment and the J _H segment from human germline JH5b. The alignment of the 47G4 V _H sequence and the germline V _H 1-69 sequence is shown in FIG. Further analysis of the 47G4 V _H sequence using the Kabat CDR region determination is shown in FIGS. 2A and 9 and in the heavy chain CDR1, CDR2 and CD3 regions as shown in SEQ ID NOs: 17, 24 and 31, respectively. It led to depiction.

47G4 Comparison of light chain immunoglobulin sequence to the known human germline immunoglobulin light chain sequences, 47G4 light chain used JK segment from V _L segments and the human germline JK3 from human germline V _K A27 Proved that. The alignment of the 47G4 V _L sequence and the germline V _K A27 sequence is shown in FIG. Further analysis of the 47G4 _VL sequence using the Kabat CDR region determination is shown in FIGS. 2B and 17 and in the light chain CDR1, CDR2 and CD3 regions as shown in SEQ ID NOs: 38, 45 and 53, respectively. It led to depiction.

  The nucleotide and amino acid sequences of the heavy chain variable region of 27F3 are shown in FIG. 3A and are shown in SEQ ID NOs: 61 and 3, respectively.

  The nucleotide and amino acid sequences of the 27F3 light chain variable region are shown in FIG. 3B and are shown in SEQ ID NOs: 69 and 11, respectively.

Comparison of 27F3 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequence shows that 27F3 heavy chain is derived from V _H segment derived from human germline V _H 5-51, derived from human germline 6-19 We have demonstrated the use of D segments and J _H segments derived from human germline JH6b. The alignment of the 27F3 V _H sequence with the germline V _H 5-51 sequence is shown in FIG. Further analysis of the 27F3 _VH sequence using the Kabat CDR region determination is shown in FIGS. 3A and 10 and for the heavy chain CDR1, CDR2 and CD3 regions as shown in SEQ ID NOs: 18, 25 and 32, respectively. It led to depiction.

Comparison of the 27F3 light chain immunoglobulin sequence with the known human germline immunoglobulin light chain sequence shows that the 27F3 light chain contains a _VL segment derived from human germline V _K L18 and a J _K segment derived from human germline JK2. It has been demonstrated to be used. An alignment of the 27F3 V _L sequence and the germline V _K L18 sequence is shown in FIG. Further analysis of the 27F3 _VL sequence using the Kabat CDR region determination is shown in FIGS. 3B and 18 and the CDR1 region, CDR2 region and CD3 region of the light chain as shown in SEQ ID NOs: 39, 46 and 54, respectively. It led to depiction.

  The nucleotide and amino acid sequences of the heavy chain variable region of 3C10 are shown in FIG. 4A and are shown in SEQ ID NOs: 62 and 4, respectively.

  The nucleotide and amino acid sequences of the light chain variable region of 3C10 are shown in FIG. 4B and are shown in SEQ ID NOs: 70 and 12, respectively.

A comparison of 3C10 heavy chain immunoglobulin sequences with known human germline immunoglobulin heavy chain sequences shows that 3C10 heavy chains are derived from human germline V _H 1-69 V _H segments, from human germline 1-26 We have demonstrated the use of D segments and J _H segments derived from human germline JH6b. An alignment of the 3C10 V _H sequence and the germline V _H 1-69 sequence is shown in FIG. Further analysis of the 3C10 V _H sequence using the Kabat CDR region determination shows that the heavy chain CDR1 region, CDR2 region and CD3 region as shown in FIGS. 4A and 11 and SEQ ID NOs: 19, 26 and 33, respectively. It led to depiction.

3C10 Comparison of light chain immunoglobulin sequence to the known human germline immunoglobulin light chain sequences, 3C10 light chain used JK segment from V _L segments and the human germline JK2 from human germline V _K L15 Proved that. An alignment of the 3C10 V _L sequence and the germline V _K L15 sequence is shown in FIG. Further analysis of the 3C10 _VL sequence using the Kabat CDR region determination is shown in FIGS. It led to depiction.

  The nucleotide and amino acid sequences of the heavy chain variable region of 5G7 are shown in FIG. 5A and are shown in SEQ ID NOs: 63 and 5, respectively.

  The nucleotide and amino acid sequences of the light chain variable region of 5G7 are shown in FIG. 5B and are shown in SEQ ID NOs: 71 and 13, respectively.

Comparison of 5G7 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequence shows that 5G7 heavy chain is derived from V _H segment derived from human germline V _H 5-51, derived from human germline 3-10 We have demonstrated the use of D segments and J _H segments derived from human germline JH6b. An alignment of the 5G7 V _H sequence and the germline V _H 5-51 sequence is shown in FIG. Further analysis of the 5G7 V _H sequence using Kabat CDR region determination is shown in FIGS. 5A and 12 and the heavy chain CDR1 region, CDR2 region and CD3 region as shown in SEQ ID NOs: 20, 27 and 34, respectively. It led to depiction.

5G7 Comparison of light chain immunoglobulin sequence to the known human germline immunoglobulin light chain sequences, 5G7 and J _K segment from V _L segments and the human germline JK1 from the light chain is the human germline V _K L18 It has been demonstrated to be used. An alignment of the 5G7 V _L sequence and the germline V _K L18 sequence is shown in FIG. Further analysis of the 5G7 _VL sequence using Kabat CDR region determination is shown in FIGS. 5B and 20 and in the light chain CDR1, CDR2 and CD3 regions as shown in SEQ ID NOs: 41, 48 and 56, respectively. It led to depiction.

  The nucleotide and amino acid sequences of the heavy chain variable region of 13F1 are shown in FIG. 6A and are shown in SEQ ID NOs: 64 and 6, respectively.

  The nucleotide and amino acid sequences of the light chain variable region of 13F1 are shown in FIG. 6B and are shown in SEQ ID NOs: 72 and 14, respectively.

Comparison of 13F1 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequence shows that 13F1 heavy chain is derived from human germline V _H 5-51 V _H segment, human germline 6-19 We have demonstrated the use of D segments and J _H segments derived from human germline JH6b. The alignment of the 13F1 V _H sequence with the germline V _H 5-51 sequence is shown in FIG. Further analysis of the 13F1 _VH sequence using the Kabat CDR region determination is shown in FIGS. 6A and 13 and the heavy chain CDR1, CDR2 and CD3 regions as shown in SEQ ID NOs: 21, 28 and 35, respectively. It led to depiction.

13f1 Comparison of light chain immunoglobulin sequence to the known human germline immunoglobulin light chain sequences, 13f1 and J _K segment from V _L segments and the human germline JK2 from the light chain is the human germline V _K L18 It has been demonstrated to be used. An alignment of the 13F1 V _L sequence and the germline V _K L18 sequence is shown in FIG. Further analysis of the 13F1 _VL sequence using the Kabat CDR region determination is shown in FIGS. 6B and 21 and in the light chain CDR1, CDR2 and CD3 regions as shown in SEQ ID NOs: 42, 49 and 57, respectively It led to depiction.

  The nucleotide and amino acid sequences of the heavy chain variable region of 46E8 are shown in FIG. 7A and are shown in SEQ ID NOs: 65 and 7, respectively.

  The nucleotide and amino acid sequences of the light chain variable region of 46E8 are shown in FIG. 7B and are shown in SEQ ID NOs: 73 and 15, respectively.

Comparison of 46E8 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequence shows that 46E8 heavy chain is derived from human germline _VH 5-51 V _H segment, human germline 6-19 We have demonstrated the use of D segments and J _H segments derived from human germline JH6b. The alignment of the 46E8 V _H sequence with the germline V _H 5-51 sequence is shown in FIG. Further analysis of the 46E8 _VH sequence using the Kabat CDR region determination is shown in FIGS. 7A and 14 and the heavy chain CDR1 region, CDR2 region and CD3 region as shown in SEQ ID NOs: 22, 29 and 36, respectively. It led to depiction.

46E8 Comparison of light chain immunoglobulin sequence to the known human germline immunoglobulin light chain sequences, 46E8 and J _K segment from V _L segments and the human germline JK2 from the light chain is the human germline V _K L18 It has been demonstrated to be used. The alignment of the 46E8 V _L sequence and the germline V _K L18 sequence is shown in FIG. Further analysis of the 46E8 _VL sequence using the Kabat CDR region determination is shown in FIGS. 7B and 22 and for the light chain CDR1, CDR2 and CD3 regions as shown in SEQ ID NOs: 43, 50 and 58, respectively. It led to depiction.

Example 3 : Characterization of binding specificity and binding kinetics of anti-CD19 human monoclonal antibodies In this example, the binding specificity of anti-CD19 antibodies 21D4 and 47G4 was tested by ELISA analysis.

Binding specificity microtiter plates by ELISA were coated with 50 μl of purified full-length CD19-Fc fusion protein at 1.0 μg / ml in PBS and then blocked with 150 μl of 1% bovine serum albumin in PBS. The plate was incubated for 30 minutes to 1 hour and washed 3 times. A dilution of HuMAb anti-CD19 antibody 47G4 was added to each well and incubated for 1 hour at 37 ° C. A known mouse anti-CD19 antibody was used as a positive control. Plates were washed with PBS / Tween and then incubated with horseradish peroxidase-conjugated goat anti-human IgG kappa specific secondary reagent for 1 hour at 37 ° C. After washing, the plate was developed with ABTS substrate (1.46 mMol / L) and analyzed at OD 490 nm. The results are shown in FIG. CD19 HuMAb 47G4 specifically bound to human CD19 peptide.

Epitope classification of anti-CD19 HuMAb was determined using epitope mapping flow cytometry of anti-CD19 antibody . Anti-CD19 human monoclonal antibodies 21D4, 21D4a, 3C10, 5G7, 5G7-N19K, 5G7-N19Q, and 13F1 binding to Raji B with either 0.3 μg / ml biotinylated 21D4 or 21D4a anti-CD19 human monoclonal antibody. Tumor cells were incubated and washed before evaluation by adding non-radioactive anti-CD19 human monoclonal antibody. An isotype control antibody was used as a negative control. Binding was detected using FITC-labeled anti-human IgG Ab. Flow cytometry analysis was performed using FACScan flow cytometry (Becton Dickinson, San Jose, CA). The results are shown in FIG. As far as this data is analyzed, anti-CD19 antibodies 21D4, 21D4a, 3C10, 5G7 and 13F1 compete for the same epitope region.

Example 4 : Binding of CD19 antibody to a B cell-derived tumor cell line
Binding of CD19 HuMAb to B cell tumor lines Raji and Daudi or to CHO-CD19 transfected cell line was assessed by flow cytometry. CHO cells were transfected with an expression plasmid containing a full-length cDNA encoding the transmembrane form of CD19. Raji, Daudi and CD19-CHO cell lines were incubated with one of the following CD19 HuMAbs, 21D4, 21D4a, 47G4, 5G7, 5G7-N19K, 5G7-N19Q, 3C10 or 13F1. A known mouse anti-CD19 antibody was used as a positive control. Cells were washed and detected with either phycoerythrin labeled anti-human or anti-mouse secondary antibodies and analyzed by flow cytometry. The results of binding experiments for the CHO-CD19 cell line, the Daudi B cell line, the Raji B cell line and the enlarged version for the Raji B cell line are shown in FIGS. 25A, 25B, 25C and 25D, respectively. Human anti-CD19 monoclonal antibodies 21D4 and 47G4 bound to the CHO-CD19 cell line. Human anti-CD19 monoclonal antibodies 21D4, 21D4a, 47G4, 5G7, 5G7-N19K, 5G7-N19Q, 3C10 and 13F1 bound to the Raji B cell line. The EC ₅₀ values of anti-CD HuMAb antibodies 21D4, 21D4a, 3C10, 5G7, 5G7-N19K, 5G7-N19Q and 13F1 were calculated as 0.1413, 0.1293, 0.2399, 0.1878, 0.2240, 0.2167 and 0.2659, respectively. 47G4 also showed binding to the Daudi B tumor cell line. All results show those measured by the geometric mean fluorescence intensity (GMFI) of staining. These data indicate that CD19 protein is expressed on the surface of B cell-derived tumor cell lines and that anti-CD19HuMAb antibodies 21D4, 21D4a, 47G4, 5G7, 5G7-N19K, 5G7-N19Q, 3C10 and 13F1 are on the cell surface It shows that it binds to CD19 expressed in FIG.

Example 5 : Scatchard binding analysis of anti-CD19 human antibodies 21D4 and 47G4 against Raji B tumor cells
Raji cells were obtained from ATCC (ATCC accession number CCL-86) and cultured in RPMI containing 10% fetal bovine serum (FBS). Cells were washed twice with 4 ° C RPMI containing 10% FBS and adjusted to 4 x 10 ⁷ cells / ml in RPMI medium containing 10% fetal calf serum (24 mM Tris pH 7.2, Binding buffer containing 137 mM NaCl, 2.7 mM KCl, 2 mM glucose, 1 mM CaCl ₂ , 1 mM MgCl ₂ , 0.1% BSA). Millipore plates (MAFB NOB) were coated with 1% skim milk in water and stored overnight at 4 ° C. The plate was washed with binding buffer and 25 μl of binding buffer containing unlabeled antibody (1000 fold excess) was added to control wells of a Millipore 96 well glass fiber filter plate (non-specific binding NSB). Only 25 microliters of buffer was added to the maximum binding control wells (total binding). Binding buffer containing 25 μl of different concentrations of ¹²⁵ I-anti-CD19 antibody 21D4 or 47G4 and 25 μl of Raji cells (4 × 10 ⁷ cells / ml) was added. The plate was incubated for 2 hours at 4 ° C. on a shaker (200 RPM). When the incubation was complete, the Millipore plate was washed three times with 0.2 ml cold wash buffer (24 mM Tris pH 7.2, 500 mM NaCl, 2.7 mM KCl, 2 mM glucose, 1 mM CaCl ₂ , 1 mM MgCl ₂ , 0.1% BSA). The filter was removed and counted with a gamma counter. Evaluation of binding equilibrium was performed with prism software (San Diego, CA) using single site binding parameters. By the Scatchard binding assay, K _D of the antibody for Raji cells, for 21D4 was approximately 2.14nM, 12.02nM for 47G4.

Example 6 : Internalization of anti-CD19 monoclonal antibody
Anti-CD19 HuMAbs were tested for their ability to internalize into Raji B tumor cells expressing CD19 or human CHO cells transfected with CD19 using the Hum-Zap internalization assay. Hum-Zap tests the internalization of primary human antibodies through the binding of secondary antibodies with affinity for human IgG bound to the toxin saporin.

The CHO-CD19 cell line or Raji B tumor cell line was seeded in 100 μl wells overnight or the next day for 2 hours at 1.0 × 10 ⁴ cells / well. Either anti-CD19 antibody 21D4 or 47G4 was added to the wells at a starting concentration of 30 nM and titrated to 1: 3 serial dilution. A human isotype control antibody non-specific for CD19 was used as a negative control. Hum-Zap (Advanced Targeting Systems, IT-22-25) was added at a concentration of 11 nM and the plates were incubated for 48 hours. The plate was then pulsed with 1.0 μCi of ³ H-thymidine for 18-24 hours, collected and read on a top count scintillation counter (Packard Instruments). The results of internalization in CHO-CD19 and B tumor cells are shown in FIGS. 26A and 26B, respectively. Only HuMAb 47G4 was tested in CHO-CD19 cells. The anti-CD19 antibody 47G4 showed an antibody concentration-dependent decrease in ³ H-thymidine incorporation in CHO-CD19 cells. Both 21D4 and 47G4 HuMAbs showed an antibody concentration-dependent decrease in ³ H-thymidine incorporation in Raji B tumor cells. This data demonstrates that the anti-CD19 antibodies 21D4 and 47G4 are internalized into CHO-CD19 transfected cells and B tumor cells that express CD19.

Example 7 : Evaluation of cell killing power of toxin-conjugated anti-CD19 antibodies In this example, anti-CD19 monoclonal antibodies conjugated to toxin were tested for their ability to kill CD19 + cell lines in a thymidine incorporation assay.

Anti-CD19 monoclonal antibody was conjugated to the toxin via a linker, such as a peptidyl linker, a hydrazone linker or a disulfide linker. Raji cell lines expressing CD19 + were seeded for 3 hours to 2.5 × 10 ⁴ cells / well. Anti-CD19 antibody-toxin complex was added to the wells at an initial concentration of 30 nM and titrated to 1: 3 serial dilution. An isotype control antibody non-specific for CD19 was used as a negative control. A 10-fold excess of either non-radioactive antibody, 21D4 or isotype control antibody was used for binding competition. Plates were incubated for 69 hours. The plate was then pulsed with 1.0 μCi of ³ H-thymidine for 24 hours, collected and read on a top count scintillation counter (Packard Instruments, Meriden, CT). The results are shown in FIG. 27 together with their EC50 values. This data demonstrates that anti-CD19 antibody 21D4 kills Raji B cell tumor cells.

Example 8 : Treatment of in vivo B cell tumor with anti-CD19 antibody In this example, SCID mice transplanted with a cancerous B cell tumor were in vivo unmodified (anti-CD19 antibody or toxin-conjugated anti-CD19 antibody). In vivo effects of antibodies on tumor growth were tested.

  Toxin-conjugated anti-CD19 antibody was prepared as described above. Severe combined immunodeficiency (SCID) mice lacking functional B and T lymphocytes were used to study B cell malignancies. Cells from the Ramos B tumor cell line were injected intravenously. The mice were treated with either 19.6 mg / kg toxin-conjugated anti-CD19 antibody or 30 mg / kg unmodified anti-CD19 antibody. An isotype control antibody or formulation buffer was used as a negative control. The animals received approximately 200 μl of PBS containing antibody or vehicle by intraperitoneal injection. The antibody-toxin conjugate is injected as a single dose on day 7 and the unmodified antibody as a single dose on day 1 for the prophylactic model or on days 7, 14 and 21 for the treatment model. Either injection of the day was given. The mice were observed daily for hind leg paralysis for approximately 6 weeks. The tumor was measured three-dimensionally using an electronic caliper (height × width × length), and the volume of the tumor was calculated. Mice were euthanized when hind leg paralysis occurred.

  Treatment with a toxin-conjugated anti-CD19 antibody, an unmodified anti-CD19 antibody administered prophylactically, or an anti-CD19 antibody administered as a therapeutic measure increases survival time as demonstrated by Kaplan-Meier analysis (Figure 28) did. The greatest increase in the indicated mean survival time was due to prophylactic treatment with unmodified anti-CD19 antibody.

  The change in body weight was also measured and calculated as a percent change in body weight. This data is shown in FIG. After 30 days, one toxin-binding antibody caused a net increase in body weight, and antibody and toxin (not complex) resulted in a net loss. Either prophylactic treatment with unmodified anti-CD19 antibody or anti-CD19 antibody treatment resulted in a net gain change.

Example 9: Treatment of in vivo tumor xenograft model with unmodified anti-CD19 antibody Mice transplanted with lymphoma tumors were treated in vivo with unmodified anti-CD19 antibody to test the in vivo effect of the antibody on tumor growth. .

ARH-77 (human B lymphoblastic leukemia; ATCC accession number CRL-1621) cells and Raji (human B lymphocytic Burkitt lymphoma; ATCC accession number CCL-86) cells were tested using standard experimental procedures. And expanded in vitro. 5 × 10 ⁶ ARH-77 cells in 0.2 ml PBS / Matrigel (1: 1) per mouse subcutaneously on the right side of 6-8 week old male CB17.SCID mice (Taconic, Hudson, NY) or Raji cells were transplanted. Twice weekly after transplantation, mice were weighed and tumors were measured three-dimensionally using an electronic caliper. Tumor volume was calculated as height x width x length / 2. Mice with an average 80 mm ³ ARH-77 tumor or an average 170 mm ³ Raji tumor were randomly added to the treatment group. On day 0, these mice received PBS vehicle, isotype control antibody or unmodified anti-CD19 HuMAb 2H5 intraperitoneally. When the tumor reached the tumor endpoint (2000 mm ³ ), the mouse was euthanized. The results are shown in FIGS. 30A (ARH-77 tumor) and 30B (Raji tumor). Unmodified anti-CD19 antibody 21D4 prolonged the mean time to reach the tumor endpoint volume (2000 mm ³ ) and slowed the progression of tumor growth. Thus, treatment with anti-CD19 antibody alone has a direct in vivo inhibitory effect on tumor growth.

Example 10 : Production of non-fucosylated HuMAbs It has been demonstrated that decreasing the amount of fucosyl residues in an antibody increases the ADCC ability of the antibody. In this example, anti-CD19 HuMAb 21D4 lacking fucosyl residues was produced.

  The CHO cell line Ms704-PF lacking the fucosyltransferase gene FUT8 (Biowa, Inc., Princeton, NJ) was electroporated using a vector expressing the heavy and light chains of antibody 21D4. Drug resistant clones were selected by culturing in Ex-Cell 325-PF CHO medium (JRH Biosciences, Lenexa, KS) containing 6 mM L-glutamine and 500 μg / ml G418 (Invitrogen, Carlsbad, Calif.). Clones were screened for IgG expression by standard ELISA methods.

Characterization of MAb oligosaccharides by CE-LIF
Comparative analysis of anti-CD19 antibody-derived N-linked oligosaccharides derived from CHO fucosylated cell lines and Ms704-PF-derived anti-CD19 monoclonal antibody samples was performed by capillary electrophoresis laser-induced fluorescence (cLIF) (Chen and Evangelista (1998) Electrophoresis 15: 1892). Purified antibody N-linked oligosaccharides were released from IgG samples (100 μg) by incubating the samples with 12.5 mU PNGaseF (Prozyme) at 40 ° C. overnight. Under the conditions used, N-linked glycans were released from the Fc portion of HuMAb 21D4 expressed in CHO and non-fucosylated cells. After removing the MAb protein by ethanol precipitation, the glycan-containing supernatant is dried by vacuum centrifugation and mild reductive amination conditions (15% in THF) to minimize desialation and loss of fucose residues. Resuspended in 19 mM 8-aminopyrene-1,3,6-trisulfonate (APTS) (Beckman) under acetic acid and 1M sodium cyanoborohydride (Sigma)). The glycan labeling reaction was continued overnight at 40 ° C., after which the sample was diluted 25-fold with water. APTS-labeled glycans were subjected to laser-induced fluorescence detection capillary electrophoresis with a P / ACE MDQ CE system (Beckman) under reverse polarity using a 50 μm ID N-CHO coated capillary (Beckman) with an effective length of 50 cm. . The sample was injected with pressure (8 seconds), and separation was carried out at 20 ° C. and 25 kV for 20 minutes using a Carbohydrate Separation Gel Buffer (Beckman). Separation was observed using a 3 mW argon ion laser and a laser-induced fluorescence detection system (Beckman) with an excitation wavelength of 488 nm and an emission wavelength of 520 nm (Ma and Nashabeh (1999) Anal. Chem. 71: 5185). Differences in oligosaccharide profiles were observed between antibodies from fucosylated cell lines and Ms704-PF cell lines. This difference was consistent with the absence of fucose residues in the anti-CD19 antibody from Ms704-PF.

Monosaccharide analysis by HPLC using HPAE-PAD
IgG samples (200 μg) were subjected to acid hydrolysis for 4 hours at 100 ° C. using either 2N TFA (when neutral sugars were expected) or 6N HCl (when amino sugars were expected). Samples were dried by vacuum centrifugation at ambient temperature and reconstituted with 200 μl water prior to analysis by HPAE-PAD (Dionex). Monosaccharides were separated using a CarboPac PA10 4 × 250 mm column with the precolumn amino trap and borate trap (Dionex). The procedure followed Dionex Technical Note 53. Monosaccharide peak type and relative abundance were determined using the monosaccharide standard (Dionex).

  Nonfucosylated anti-CD19 21D4 antibody was also tested using a standard capillary isoelectric focusing kit assay (Beckman Coulter). From this assay, pI values of pH 8.45 for fucosylated 21D4 antibody, pH 8.44 and 8.21 for fucosylated 21D4a antibody, and pH 8.52 and 8.30 for non-fucosylated 21D4 antibody were observed.

Example 11 : Evaluation of ADCC activity of anti-CD19 antibody In this example, the ability to kill CD19 + cells in the presence of effector cells via antibody-dependent cytotoxicity (ADCC) in a fluorescent cytotoxicity assay. Were tested for fucosylated and non-fucosylated anti-CD19 monoclonal antibodies.

Nonfucosylated human anti-CD19 monoclonal antibody 21D4 was prepared as described above. Human effector cells were prepared from whole blood as follows. Human peripheral blood mononuclear cells were purified from heparinized whole blood by standard Ficoll-paque separation. These cells were resuspended in RPMI 1640 medium containing 10% FBS (medium) and 200 U / ml human IL-2 and incubated overnight at 37 ° C. The next day cells were harvested, washed once with media and resuspended to 2 × 10 ⁷ cells / ml. Target CD19 + cells were incubated with BATDA reagent (Perkin Elmer, Wellesley, MA) (2.5 μl BATDA per 1 × 10 ⁶ target cells / mL in 2.5 mM probenecid supplemented medium (assay medium)) at 37 ° C. for 20 minutes. Target cells were washed 4 times with PBS containing 20 mM HEPES and 2.5 mM probenecid and spun down to a final volume of 1 × 10 ⁵ cells / ml in assay medium.

  CD19 + cell line ARH-77 (human B lymphoblastic leukemia; ATCC accession number) for antibody-specific ADCC against fucosylated and nonfucosylated human anti-CD19 monoclonal antibody 21D4 using Delfia fluorescence analysis as follows: CRL-1621) was tested. Target cell line ARH77 (100 μl labeled target cells) was incubated with 50 μl effector cells and either 50 μl 21D4 antibody or non-fucosylated 21D4 antibody. Throughout this experiment, a 1:50 target: effector ratio was used. A human IgG1 isotype control was used as a negative control. After 2100 rpm pulsed spin and incubation at 37 ° C. for 1 hour, the supernatant was collected, spun quickly again, 20 μl of the supernatant was transferred to a flat bottom plate, and 180 μl of Eu solution (Perkin Elmer, Wellesley, MA) ) And read with a Fusion Alpha TRF plate reader (Perkin Elmer). The% dissolution was calculated from the following formula: (sample release-spontaneous release * 100) / (maximum release-spontaneous release). Where spontaneous release is fluorescence from wells containing only target cells and maximal release is fluorescence from wells containing target cells and treated with 3% Lysol. The% cytotoxic activity specific lysis for the ARH-77 cell line is shown in FIG. The CD19 + expressing cell line ARH-77 showed increased antibody-mediated cytotoxic activity by the HuMAb anti-CD19 antibody 21D4 and an increased rate of specific lysis associated with the non-fucosylated form of the anti-CD19 antibody 21D4. This data demonstrates that the non-fucosylated HuMAb anti-CD19 antibody exhibits increased specific cytotoxic activity against CD19 + expressing cells.

Example 12 : Thermal stability of anti-CD19 monoclonal antibodies by differential scanning calorimetry The thermal stability of anti-CD19 monoclonal antibodies was compared using calorimetric analysis of their melting temperatures.

  Calorimetry (registered trademark) of the melting temperature was performed using a VP-capillary DSC differential scanning microcalorimeter platform (MicroCal LLC, Northampton, MA, USA) connected to an automatic sampler. The sample cell volume is 0.144 mL. Denaturation data for the glycosylated and deglycosylated forms of the antibody were obtained by heating a sample at a concentration of 2.3 μM between 30-95 ° C. at a rate of 1 ° C./min. Protein samples were placed in phosphate buffered saline (PBS) at pH 7.4. The same buffer was used in the reference cells and the molar heat capacity was obtained by comparison. The observed thermograms were baseline corrected and normalized data was analyzed based on the two-state model using the software Origin v.7.0. This data is shown in Table 2.

(Table 2) Measurement of thermal stability of anti-CD19 antibody

Example 13 : Evaluation of glycosylation sites
HuMAb anti-CD19 antibody 5G7 was found by sequence analysis to have an NXS / T glycosylation motif in its variable region. The presence of N-linked sequence sites (NXS / T) is necessary but not sufficient for the addition of saccharides to MAbs. That is, it may have an NXS / T sequence that does not actually add sugars due to protein folding and solvent accessibility. Confirmation of glycosylation sites in the variable region of 5G7 was tested by both LC-MS and Western analysis.

  Liquid chromatography-mass spectrometry (LC-MS) is a standard tool for determining the mass of proteins such as antibodies. Anti-CD19 HuMAb 5G7 and 13F1 N-linked oligosaccharides were released from IgG samples (100 μg) by incubating the samples with 12.5 mU PNGaseF (Prozyme) overnight at 40 ° C. prior to analysis. Under the conditions used, N-linked glycans were released from the Fc portion of HuMAb. For clone 5G7, we observed two masses of high abundance; one mass (49,855 Da) is after removal of the sugar at the conserved N-binding site (N297) of the constant region by PNGaseF digestion. It corresponds to the estimated mass, and the second mass (52,093 Da) corresponds to the saccharide added to the second site. We find that Fab region glycans are not removed by PNGaseF digestion; therefore, this data supports the presence of sugars in the variable region of clone 5G7. For clone 13F1, the observed mass was consistent with the estimated mass of the protein sequence to which no saccharide was added.

  To confirm the above results, we performed a Western blot assay for the Fab fragments of clones 5G7 and 13F1 using a saccharide specific staining method. Fab and Fc fragments were obtained by adding 1.25 μg activated papain to a 25 μg IgG sample containing 1 mM cysteine. Samples were incubated at 40 ° C. for 4 hours and the reaction was stopped with 30 mM iodoacetamide. Samples were analyzed by 4-20% Tris-Glycine SDS-PAGE followed by electroblotting to PVDF membrane. Saccharide-specific staining of this blot was performed using a Gel Code Glycoprotein Staining Kit (Pierce) according to the protocol specified by the manufacturer. The results detected Fab glycosylation in the 5G7 antibody, but not in the 13F1 antibody. These results indicated that the 5G7 antibody is glycosylated in the Fab region.

  As described above, anti-CD19 monoclonal antibody 5G7 contains a variable region with a glycosylation site. Variable region glycosylation sites increase antibody immunogenicity and can alter pK values by altering antigen binding, thus mutating variable region NXS / T glycosylation motif sequences to reduce glycosylation It may be advantageous to do so. Using standard molecular biology techniques, the 5G7 antibody sequence was modified to change the N-I-S sequence starting at position 19 to either K-I-S (5G7-N19K) or Q-I-S (5G7-N19Q).

Example 14 Stability of Anti-CD19 Monoclonal Antibody by Fluorescence Spectroscopy The stability of anti-CD19 monoclonal antibody was compared by measuring the midpoint of chemical denaturation by fluorescence spectroscopy.

  Chemical denaturation fluorescence measurements were performed using SPEX Fluorolog 3.22 (SPEX, Edison, NJ) with a Micromax plate reader. Measurements were performed using antibody samples that were allowed to stand for 24 hours in PBS buffer containing 16 different concentrations of guanidinium hydrochloride. Measurements were performed in black, low volume, non-binding surface 384 well plates (Corning, Acton, Mass.), Requiring 2 μM antibody in a 12 μL well volume. The fluorescence was excited at 280 nm and the emission spectrum was measured from 300 to 400 nm. The scanning speed was 1 second / nm, and the slit was set in the 5 nm band. Buffer blanks were made with PBS and automatically subtracted from the data. The data is shown in Table 3.

(Table 3) Fluorescence stability of anti-CD19 antibody

Example 15 : Treatment of in vivo B cell Raji tumors with anti-CD19 antibodies In this example, SCID mice transplanted with cancerous B cell tumors were treated with either unmodified anti-CD19 antibodies or toxin-conjugated anti-CD19 antibodies. Treated in vivo and tested the in vivo effect of the antibody on tumor growth.

Toxin-conjugated anti-CD19 antibody was prepared as described above. Severe combined immunodeficiency (SCID) mice lacking functional B and T lymphocytes were used to study B cell malignancies. Cells from Raji B tumor cell line were injected subcutaneously. The mice were treated with 30 mg / kg antibody or 0.3 μmol / kg (toxin) antibody-toxin conjugate. An isotype control antibody or formulation buffer was used as a negative control. The animals received approximately 200 μl of PBS containing antibody or vehicle by intraperitoneal injection. The antibody was either injected on day 0 as a single dose (SD) or injected on days 0, 7 and 14 as repeated doses (RD). The mice were observed daily for tumor growth using an electronic caliper; tumors were measured in three dimensions (height x width x length / 2) and tumor volume was calculated. Mice were euthanized when the tumor reached the tumor endpoint (2000 mm ³ ) or showed weight loss greater than 20%. The results are shown in FIG. In each case, the anti-CD19 antibody showed a smaller tumor volume compared to the negative control, and the toxin-bound antibody showed a smaller tumor volume than treatment with the unmodified antibody.

  Changes in body weight were also measured and calculated as% change in body weight. The results are shown in FIG. The results showed that body weight changed purely when using toxin-conjugated antibodies and increased when using either excipients or unmodified antibodies.

Example 16 : B cell studies in cynomolgus monkeys In this example, either parental anti-CD19 antibodies or non-fucosylated (nf) anti-CD19 antibodies were injected intravenously into cynomolgus monkeys. Absolute counts of leukocytes and a subset of leukocytes were determined after administration and compared to pre-dose values.

  Blood samples taken from cynomolgus monkeys were stained with either parental CD19 antibody or nf anti-CD19 antibody and analyzed by FACS using standard methods. All monkey B cells used in this study were positively stained with both parental and nf anti-CD19 antibodies. Each group included 2 male and 2 female monkeys. Blood samples were collected on day 7 and before dosing. Bolus intravenous injections were made slowly into the saphenous vein and animals were administered 1 mg / kg parent or nf anti-CD19 antibody. Blood samples were collected 24 hours, 48 hours, 72 hours, and 7 days, 14 days, 21 days and 28 days after administration. Blood samples were taken for PK determination, hematology and for flow cytometry. At each time point, the following cell surface antigens were observed from the blood: CD2 + / CD20 + (all lymphocytes), CD20 + (B lymphocytes), CD3 + (T lymphocytes), CD3 + / CD4 + (T helper lymphocytes), CD3 + / CD8 + (T cytotoxic lymphocytes), CD3- / CD16 + (NK cells), CD3- / CD14 + (monocytes).

  FIG. 34 shows the change in the number of CD20 positive cells when compared to the mean value before day 7 and before administration. The parent anti-CD19 antibody induced a 55% decrease in the number of CD20 positive B cells after 24 hours, whereas the nonfucosylated antibody resulted in a more potent B cell number suppressive decrease of approximately 90%. In the nf anti-CD19 group, the B cell count remained at this level after 2, 3, and 7 days of treatment, but the parent antibody was apparently beginning to return to baseline. FIG. 35 shows the% change from the baseline for CD20 positive cells in each individual monkey. All four monkeys treated with nf anti-CD19 antibody showed a greater reduction in% of CD20 positive cells compared to the parent anti-CD19 antibody. Taken together, these data suggest that the nf anti-CD19 antibody is more effective at depleting circulating B cells compared to its parent antibody.

Example 17 : Immunohistochemical study of anti-CD19 antibody
To assess the tissue binding profile of HuMab anti-CD19, FITC-conjugated 21D4 (21D4-FITC, F: P = 4) and non-fucosylated 21D4 (nf21D4) (nf21D4-FITC, F: P = 3), spleen, Normal (non-neoplastic) human tissues including tonsils, small intestine, cerebellum, cerebrum, heart, liver, lung and kidney (1-2 samples each) and chronic lymphocytic leukemia, follicular lymphoma, marginal layer lymphoma, mantle Tested with a panel of B cell neoplasms (1-2 samples each), including cellular lymphoma and diffuse large B cell lymphoma. Non-fucosylated 21D4 antibody was prepared as described above. FITC-conjugated Hu-IgG ₁ (Hu-IgG ₁ -FITC) was used as an isotype control antibody.

  Normal and lymphoma tissues snap frozen and embedded in OCT were purchased from the Cooperative Human Tissue Network (Philadelphia, PA) or National Disease Research Institute (Philadelphia, PA). 5 μm cryostat sections were fixed with acetone for 10 minutes at room temperature and stored at −80 ° C. until use. Indirect peroxidase immunostaining using EnVision + System (Dako. Carpinteria, CA) was performed according to our routine protocol. Briefly, slides were washed twice with PBS (Sigma, St. Louis, MO) and then incubated for 10 minutes with the peroxidase block provided as Dako EnVision + System. After washing twice with PBS, nonspecific binding sites were blocked by incubating slides with Dako protein block supplemented with 1% human gamma globulin and 1 mg / ml heat-aggregated human IgG for 20 minutes. The primary antibody (0.4 μg / ml or 2 μg / ml 21D4-FITC and nf21D4-FITC) or isotype control (0.4 μg / ml or 2 μg / ml Hu-IgG1-FITC) is then applied to the sections for 1 hour Incubated. After washing three times with PBS, the slides were incubated with mouse anti-FITC antibody (20 μg / ml) for 30 minutes. After three additional washes with PBS, slides were incubated for 30 minutes with peroxidase-conjugated anti-mouse IgG polymer provided as Dako EnVision + System. Finally, slides were washed as described above and reacted with DAB substrate-chromogen solution provided as Dako EnVision + System for 6 minutes. The slides were then washed with deionized water according to conventional histological procedures, counterstained with Mayer's hematoxylin (Dako), dehydrated, washed, and Permount (Fischer Scientific, Fair Lawn, NJ) was applied as a coverslip. .

  Specific staining with both 21D4-FITC and nf21D4-FITC was observed in lymphoid tissue or lymphoid rich tissue (spleen, tonsils and small intestine) and lymphoma tissue. In the spleen and tonsil, strong specific staining was mainly distributed in the B cell region, namely the splenic lymph node, tonsil mantle zone and germinal center. In the small intestine, strong specific immune responses were mainly localized in Peyer's patches or lymphoid aggregates, and weak to strong staining was localized in lymphocytes dispersed in the lamina propria of the mucosa. Strong staining was also shown in tumor cells of follicular lymphoma and marginal layer lymphoma, and moderate to strong staining was shown in chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and mantle cell lymphoma.

  In normal cerebellum, cerebrum, heart, liver, lung, and kidney tissue, staining with either 21D4-FITC or nf21D4-FITC, some of the focal lymphoid cells or aggregates in lung tissue and kidney tissue No significant staining was observed except for staining. In addition, these tissues stained at high concentrations up to 10 μg / ml. Similarly, no specific staining was observed compared to the isotype control antibody.

  Comparison of 21D4-FITC and nf21D4-FITC showed similar staining patterns in all tissues. This specific staining was saturated or nearly saturated at 0.4 μg / ml. However, the staining intensity with 21D4-FITC is about 0.5-1 grade stronger than that with nf21D4-FITC. This is probably due in part to the high F: P ratio of 21D4-FITC (4 to 3).

Example 18 : Evaluation of cell killing power of anti-CD19 antibody In this example, the anti-CD19 monoclonal antibody was tested alone or conjugated to a toxin for its ability to kill the CD19 + cell line in a thymidine incorporation assay.

Anti-CD19 monoclonal antibody was conjugated to the toxin via a linker, such as a peptidyl linker, a hydrazone linker or a disulfide linker. Raji or SU-DHL-6 cell lines expressing CD19 + were seeded at 1 × 10 ⁴ cells / well. Either anti-CD19 antibody alone or anti-CD19 antibody-toxin conjugate was added to the wells at a starting concentration of 30 nM and titrations were performed to give 1: 3 serial dilutions for the 8 dilutions. A human isotype control antibody non-specific for CD19 was used as a negative control. Plates were incubated for 69 hours. The plate was then pulsed with 0.5 μCi of ³ H-thymidine for 24 hours, collected and read on a top count scintillation counter (Packard Instruments, Meriden, CT). The results are shown in FIG. 36 together with their EC50 values. FIG. 36A shows unmodified antibody against Raji cells. FIG. 36B shows an unmodified antibody against SU-DHL-6 cells. FIG. 36C shows a toxin-conjugated anti-CD19 antibody against SU-DHL-6 cells. This data demonstrates that anti-CD19 antibody 21D4 binds to and kills Raji B cell tumor cells and has an unexpectedly high level of cell killing power against SU-DHL-6 cells.

Example 19 : B cell depletion study A whole blood B cell depletion assay was designed to determine whether anti-CD19 antibodies can deplete B cells.

Human whole blood was purchased from AllCells Inc. (Berkeley, CA) and transferred to room temperature on the same day. 2 ml whole blood was incubated in the absence of the intended antibody or in the presence of 1-30 mg / ml, or in the presence of PBS as an untreated group. The blood-antibody mixture was incubated overnight at 37 ° C. with 5% CO ₂ . On the day of the experiment, blood was lysed twice with RBC lysis buffer at a 1:10 ratio by incubation for 10 minutes followed by centrifugation. After the second centrifugation, the cell pellet is washed once with FACS buffer (PBS plus calcium and magnesium containing 2% FBS and 20% versene) followed by a T cell marker using a standard flow cytometry protocol FACS staining was performed with an anti-CD3 antibody (Becton Dickinson catalog number 555332) and an anti-CD22 antibody (Becton Dickinson catalog number 340708) as a B cell marker. Cells were incubated on ice for 20 minutes before a final wash and resuspended in 5 mg / ml propidium iodide solution (Sigma catalog number P4864) in FACS buffer. Data were collected by flow cytometry using the FASCalibur system from Becton Dickinson and Cellquest software and analyzed using lymphocyte size gating through FlowJo software. The percent change was calculated by determining% positive B cells in the untreated group-% positive B cells in the antibody treated group /% positive B cells in the untreated group × 100. The results are shown in Table 4. 8.7% B cells from healthy blood donors were retained in the blood after overnight incubation (no antibody). Incubation of 30 mg / ml positive control Rituxan with whole blood showed a 46% depletion in B cell count when compared to the untreated antibody-free group. The group treated with non-fucosylated (nf) anti-CD19 antibody had a clear effect on B cell depletion and suppressed B cells by approximately 40%. The parent antibody had a moderate effect on B cell count.

(Table 4) B cell depletion from whole blood

Overview of sequence listing

The nucleotide sequence (SEQ ID NO: 59) and amino acid sequence (SEQ ID NO: 1) of the heavy chain variable region of the 21D4 and 21D4a human monoclonal antibodies are shown. The CDR1 (SEQ ID NO: 16), CDR2 (SEQ ID NO: 23), and CDR3 (SEQ ID NO: 30) regions are delineated and the V, D, and J germline origins are shown. 2A shows the nucleotide sequence (SEQ ID NO: 66) and amino acid sequence (SEQ ID NO: 8) of the light chain variable region of the 21D4 human monoclonal antibody. The CDR1 (SEQ ID NO: 37), CDR2 (SEQ ID NO: 44), and CDR3 (SEQ ID NO: 51) regions are delineated to indicate the origin of the V and J germline. 2A shows the nucleotide sequence (SEQ ID NO: 67) and amino acid sequence (SEQ ID NO: 9) of the light chain variable region of the 21D4a human monoclonal antibody. The CDR1 (SEQ ID NO: 37), CDR2 (SEQ ID NO: 44), and CDR3 (SEQ ID NO: 52) regions are delineated to indicate the origin of the V and J germline. The nucleotide sequence (SEQ ID NO: 60) and amino acid sequence (SEQ ID NO: 2) of the heavy chain variable region of the 47G4 human monoclonal antibody are shown. The CDR1 (SEQ ID NO: 17), CDR2 (SEQ ID NO: 24), and CDR3 (SEQ ID NO: 31) regions are delineated and the V, D, and J germline origins are shown. The nucleotide sequence (SEQ ID NO: 68) and amino acid sequence (SEQ ID NO: 10) of the light chain variable region of the 47G4 human monoclonal antibody are shown. The CDR1 (SEQ ID NO: 38), CDR2 (SEQ ID NO: 45), and CDR3 (SEQ ID NO: 53) regions are delineated to indicate the origin of the V and J germline. The nucleotide sequence (SEQ ID NO: 61) and amino acid sequence (SEQ ID NO: 3) of the heavy chain variable region of the 27F3 human monoclonal antibody are shown. The CDR1 (SEQ ID NO: 18), CDR2 (SEQ ID NO: 25), and CDR3 (SEQ ID NO: 32) regions are delineated and the V, D, and J germline origins are shown. The nucleotide sequence (SEQ ID NO: 69) and amino acid sequence (SEQ ID NO: 11) of the light chain variable region of the 27F3 human monoclonal antibody are shown. The CDR1 (SEQ ID NO: 39), CDR2 (SEQ ID NO: 46), and CDR3 (SEQ ID NO: 54) regions are delineated to indicate the origin of the V and J germline. 3A shows the nucleotide sequence (SEQ ID NO: 62) and amino acid sequence (SEQ ID NO: 4) of the heavy chain variable region of the 3C10 human monoclonal antibody. The CDR1 (SEQ ID NO: 19), CDR2 (SEQ ID NO: 26), and CDR3 (SEQ ID NO: 33) regions are delineated and the V, D, and J germline origins are shown. 3A shows the nucleotide sequence (SEQ ID NO: 70) and amino acid sequence (SEQ ID NO: 12) of the light chain variable region of the 3C10 human monoclonal antibody. The CDR1 (SEQ ID NO: 40), CDR2 (SEQ ID NO: 47), and CDR3 (SEQ ID NO: 55) regions are delineated to indicate the origin of the V and J germline. The nucleotide sequence (SEQ ID NO: 63) and amino acid sequence (SEQ ID NO: 5) of the heavy chain variable region of the 5G7 human monoclonal antibody are shown. The CDR1 (SEQ ID NO: 20), CDR2 (SEQ ID NO: 27), and CDR3 (SEQ ID NO: 34) regions are delineated and the V, D, and J germline origins are shown. The nucleotide sequence (SEQ ID NO: 71) and amino acid sequence (SEQ ID NO: 13) of the light chain variable region of the 5G7 human monoclonal antibody are shown. The CDR1 (SEQ ID NO: 41), CDR2 (SEQ ID NO: 48), and CDR3 (SEQ ID NO: 56) regions are delineated to indicate the origin of the V and J germline. The nucleotide sequence (SEQ ID NO: 64) and amino acid sequence (SEQ ID NO: 6) of the heavy chain variable region of the 13F1 human monoclonal antibody are shown. The CDR1 (SEQ ID NO: 21), CDR2 (SEQ ID NO: 28), and CDR3 (SEQ ID NO: 35) regions are delineated and the V, D, and J germline origins are shown. The nucleotide sequence (SEQ ID NO: 72) and amino acid sequence (SEQ ID NO: 14) of the light chain variable region of the 13F1 human monoclonal antibody are shown. The CDR1 (SEQ ID NO: 42), CDR2 (SEQ ID NO: 49), and CDR3 (SEQ ID NO: 57) regions are delineated to indicate the origin of the V and J germline. The nucleotide sequence (SEQ ID NO: 65) and amino acid sequence (SEQ ID NO: 7) of the heavy chain variable region of the 46E8 human monoclonal antibody are shown. The CDR1 (SEQ ID NO: 22), CDR2 (SEQ ID NO: 29), and CDR3 (SEQ ID NO: 36) regions are delineated and the V, D, and J germline origins are shown. The nucleotide sequence (SEQ ID NO: 73) and amino acid sequence (SEQ ID NO: 15) of the light chain variable region of the 46E8 human monoclonal antibody are shown. The CDR1 (SEQ ID NO: 43), CDR2 (SEQ ID NO: 50), and CDR3 (SEQ ID NO: 58) regions are delineated to indicate the origin of the V and J germline. Shows the alignment of the amino acid sequence of the heavy chain variable region of 21D4 (SEQ ID NO: 1) and 21D4a (SEQ ID NO: 1) with the amino acid sequence of human germline V _H 5-51 (SEQ ID NO: 74) . The JH4b germline is disclosed as SEQ ID NO: 80. The alignment of the amino acid sequence of the heavy chain variable region of 47G4 (SEQ ID NO: 2) and the amino acid sequence of human germline V _H 1-69 (SEQ ID NO: 75) is shown. The JH5b germline is disclosed as SEQ ID NO: 81. The alignment of the amino acid sequence of the heavy chain variable region of 27F3 (SEQ ID NO: 3) and the amino acid sequence of human germline V _H 5-51 (SEQ ID NO: 74) is shown. The JH6b germline is disclosed as SEQ ID NO: 82. The alignment of the amino acid sequence of the heavy chain variable region of 3C10 (SEQ ID NO: 4) and the amino acid sequence of human germline V _H 1-69 (SEQ ID NO: 75) is shown. The JH6b germline is disclosed as SEQ ID NO: 82. The alignment of the amino acid sequence of the heavy chain variable region of 5G7 (SEQ ID NO: 5) and the amino acid sequence of human germline V _H 5-51 (SEQ ID NO: 74) is shown. The JH6b germline is disclosed as SEQ ID NO: 83. The alignment of the amino acid sequence of the heavy chain variable region of 13F1 (SEQ ID NO: 6) and the amino acid sequence of human germline V _H 5-51 (SEQ ID NO: 74) is shown. The JH6b germline is disclosed as SEQ ID NO: 82. The alignment of the amino acid sequence of the heavy chain variable region of 46E8 (SEQ ID NO: 7) and the amino acid sequence of human germline V _H 5-51 (SEQ ID NO: 74) is shown. The JH6b germline is disclosed as SEQ ID NO: 82. 2 shows the alignment of the amino acid sequence of the light chain variable region of 21D4 (SEQ ID NO: 8) with the amino acid sequence of human germline V _K L18 (SEQ ID NO: 76). The JK2 germline is disclosed as SEQ ID NO: 84. 2 shows the alignment of the amino acid sequence of the light chain variable region of 21D4a (SEQ ID NO: 9) with the amino acid sequence of human germline V _K L18 (SEQ ID NO: 76). The JK3 germline is disclosed as SEQ ID NO: 85. 47G4 shows the alignment of:: (77 SEQ ID NO) (SEQ ID NO 10) and amino acid sequence of the light chain variable region of the amino acid sequence of the human germline V _K A27. The JK3 germline is disclosed as SEQ ID NO: 85. The alignment of the amino acid sequence of the light chain variable region of 27F3 (SEQ ID NO: 11) and the amino acid sequence of human germline V _K L18 (SEQ ID NO: 76) is shown. The JK2 germline is disclosed as SEQ ID NO: 84. 3 shows the alignment of the amino acid sequence of the light chain variable region of 3C10 (SEQ ID NO: 12) with the amino acid sequence of human germline V _K L15 (SEQ ID NO: 78). The JK2 germline is disclosed as SEQ ID NO: 84. 5 shows the alignment of the amino acid sequence of the light chain variable region of 5G7 (SEQ ID NO: 13) with the amino acid sequence of human germline V _K L18 (SEQ ID NO: 76). The JK1 germline is disclosed as SEQ ID NO: 86. The alignment of the amino acid sequence of the light chain variable region of 13F1 (SEQ ID NO: 14) and the amino acid sequence of human germline V _K L18 (SEQ ID NO: 76) is shown. The JK2 germline is disclosed as SEQ ID NO: 87. The alignment of the amino acid sequence of the light chain variable region of 46E8 (SEQ ID NO: 15) and the amino acid sequence of human germline V _K L18 (SEQ ID NO: 76) is shown. The JK2 germline is disclosed as SEQ ID NO: 87. It is a graph which shows the experimental result which demonstrates that the human monoclonal antibody 47G4 with respect to human CD19 specifically couple | bonds with human CD19. It is a graph which shows the experimental result which demonstrates that the human monoclonal antibody with respect to CD19 competes for the binding with respect to Raji cells. Shown are flow cytometry results demonstrating that human monoclonal antibodies 21D4, 21D4a, 47G4, 3C10, 5G7 and 13F1 against human CD19 bind to the cell surface of B cell tumor cell lines. Flow cytometry of HuMAb 21D4 and 47G4 on CHO cells transfected with human CD19. Shown are flow cytometry results demonstrating that human monoclonal antibodies 21D4, 21D4a, 47G4, 3C10, 5G7 and 13F1 against human CD19 bind to the cell surface of B cell tumor cell lines. Flow cytometry of HuMAb 47G4 on Daudi B tumor cells. Shown are flow cytometry results demonstrating that human monoclonal antibodies 21D4, 21D4a, 47G4, 3C10, 5G7 and 13F1 against human CD19 bind to the cell surface of B cell tumor cell lines. Flow cytometry of HuMAb 21D4 and 47G4 against Raji B tumor cells. Shown are flow cytometry results demonstrating that human monoclonal antibodies 21D4, 21D4a, 47G4, 3C10, 5G7 and 13F1 against human CD19 bind to the cell surface of B cell tumor cell lines. Flow cytometry of HuMAbs 21D4, 21D4a, 3C10, 5G7 and 13F1 against Raji B tumor cells. Figure 3 shows the results of an internalization experiment demonstrating by human 3H-thymidine release assay that human monoclonal antibodies 21D4 and 47G4 against human CD19 invade Raji B tumor cells expressing CHO-CD19 and CD19. Internalization of HuMAb 47G4 into CHO-CD19 cells. Figure 3 shows the results of an internalization experiment demonstrating by human 3H-thymidine release assay that human monoclonal antibodies 21D4 and 47G4 against human CD19 invade Raji B tumor cells expressing CHO-CD19 and CD19. Internalization of HuMAb 21D4 and 47G4 into Raji B tumor cells. Figure 3 shows the results of a thymidine incorporation assay that demonstrates that human monoclonal antibodies against human CD19 kill Raji B cell tumor cells. Figure 2 shows Kaplan-Meier plot of mouse survival in Ramos systemic kinetic model. The weight change of a mouse | mouth in a Ramos whole body dynamic model is shown. Figure 3 shows the results of an in vivo mouse tumor model study demonstrating that treatment with unmodified anti-CD19 antibody 21D4 has a direct suppressive effect on lymphoma tumors in vivo. (A) ARH-77 tumor. (B) Raji tumor. 2 shows the results of an ADCC assay demonstrating that non-fucosylated human monoclonal anti-CD19 antibodies increase cytotoxicity against human leukemia cells in an antibody dependent cellular cytotoxicity (ADCC) dependent manner. 3 shows the results of an in vivo mouse tumor model study demonstrating that toxin-conjugated anti-CD19 antibody reduces tumor volume. Figure 3 shows changes in body weight of mice in Raji tumor model studies. Figure 3 shows the results of a cynomolgus monkey study showing a decrease in the CD20 + cell population following fucosylated or non-fucosylated anti-CD19 HuMAb treatment. Shown are the results of individual cynomolgus monkeys after fucosylated or non-fucosylated anti-CD19 HuMAb treatment. 3 shows the results of a thymidine incorporation assay that demonstrates that a human monoclonal antibody alone or a toxin conjugate thereof against human CD19 kills Raji B cell tumor cells and SU-DHL-6 B cell tumor cells. 3 shows the results of a thymidine incorporation assay that demonstrates that a human monoclonal antibody alone or a toxin conjugate thereof against human CD19 kills Raji B cell tumor cells and SU-DHL-6 B cell tumor cells. 3 shows the results of a thymidine incorporation assay that demonstrates that a human monoclonal antibody alone or a toxin conjugate thereof against human CD19 kills Raji B cell tumor cells and SU-DHL-6 B cell tumor cells.

Claims (34)

(A) heavy chain CDR1 comprising an amino acid having the sequence shown in SEQ ID NO: 16; heavy chain CDR2 comprising an amino acid having the sequence shown in SEQ ID NO: 23; having the sequence shown in SEQ ID NO: 30 A heavy chain CDR3 comprising amino acids; a light chain CDR1 comprising amino acids having the sequence shown in SEQ ID NO: 37; a light chain CDR2 comprising amino acids having the sequence shown in SEQ ID NO: 44; and SEQ ID NO: 51 A light chain CDR3 comprising an amino acid having the sequence shown;
(B) heavy chain CDR1 comprising an amino acid having the sequence shown in SEQ ID NO: 16; heavy chain CDR2 comprising an amino acid having the sequence shown in SEQ ID NO: 23; having the sequence shown in SEQ ID NO: 30 A heavy chain CDR3 comprising amino acids; a light chain CDR1 comprising amino acids having the sequence shown in SEQ ID NO: 37; a light chain CDR2 comprising amino acids having the sequence shown in SEQ ID NO: 44; and SEQ ID NO: 52 A light chain CDR3 comprising an amino acid having the sequence shown; or (c) a heavy chain CDR1 comprising an amino acid having the sequence shown in SEQ ID NO: 17; a heavy chain comprising an amino acid having the sequence shown in SEQ ID NO: 24 CDR2; heavy chain CDR3 comprising an amino acid having the sequence shown in SEQ ID NO: 31; light chain CDR1 comprising an amino acid having the sequence shown in SEQ ID NO: 38; amino acid having a sequence shown in SEQ ID NO: 45 A light chain comprising the amino acid having the sequence shown in SEQ ID NO: 53 CDR3;
Including,
A monoclonal antibody or antigen-binding portion thereof that specifically binds to CD19 . (A) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO: 1; and (b) a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO: 8;
including,
The monoclonal antibody or antigen-binding portion thereof according to claim 1. (A) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO: 1; and (b) a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO: 9;
including,
The monoclonal antibody or antigen-binding portion thereof according to claim 1. (A) a heavy chain variable region comprising an amino acid having the sequence shown in SEQ ID NO: 2; and (b) a light chain variable region comprising an amino acid having the sequence shown in SEQ ID NO: 10.
including,
The monoclonal antibody or antigen-binding portion thereof according to claim 1. The monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 4 , which binds to Raji B cell tumor cells and Daudi B cell tumor cells. The monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 4 , which is a human antibody or a portion thereof. The monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 4 , which is a humanized antibody or chimeric antibody or a portion thereof. The monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 4 , wherein CD19 is human CD19, which is a polypeptide comprising an amino acid having the sequence shown in SEQ ID NO: 79. The monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 4 , which is a full-length antibody of IgG1 isotype or IgG4 isotype or a portion thereof. The monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 4 , which is a single chain antibody. The monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 4 , which is nonfucosylated. 1 × 10 ^-7 M or less binds to human CD19 with a K _D of monoclonal antibody or antigen binding portion thereof according to any of claims 1 - 4. Binds to human CD19 5 × 10 ^-8 M or or less K _D, monoclonal antibody or antigen binding portion thereof of claim 12. It binds to human CD19 with 5 × 10 ^-9 M or less K _D, monoclonal antibody or antigen binding portion thereof of claim 13. The monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 4 , which has a heat stable temperature of 65 ° C or higher. A composition comprising the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier. 16. An immune complex comprising the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-15 linked to a therapeutic agent. 18. The immune complex according to claim 17 , wherein the therapeutic agent is a cytotoxin or a radioisotope. A composition comprising the immune complex of claim 17 or 18 and a pharmaceutically acceptable carrier. An isolated nucleic acid encoding the monoclonal antibody or antigen-binding portion thereof of any one of claims 1-15 . An expression vector comprising the nucleic acid according to claim 20 . A host cell comprising the expression vector according to claim 21 . 23. A method for preparing an anti-CD19 monoclonal antibody or antigen-binding portion thereof, comprising the steps of expressing an antibody or antigen-binding portion thereof in a host cell according to claim 22 and isolating the antibody or antigen-binding portion thereof from the host cell. . In vitro of the growth of tumor cells expressing CD19 comprising contacting the cells with an amount of the monoclonal antibody or antigen binding portion thereof of any of claims 1-15 effective to inhibit the growth of tumor cells. Inhibition method. Use of the monoclonal antibody or antigen-binding portion thereof according to any of claims 1-15 for the manufacture of a medicament for inhibiting the growth of tumor cells expressing CD19 in a subject. 26. Use according to claim 25 , wherein the tumor cells are B cell malignant tumor cells. Non-Hodgkin lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, Burkitt lymphoma, undifferentiated large cell lymphoma, multiple myeloma, lymphocytic lymphoma, follicular lymphoma cancer 27. Use according to claim 26 , which is diffuse large B cell lymphoma, Castorman's disease or Waldenstrom's globulinemia. 27. Use according to claim 26 , wherein the B cell malignancy is mantle cell lymphoma. The monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 15 , which inhibits the growth of tumor cells expressing CD19. Use of the monoclonal antibody or antigen-binding portion thereof according to any of claims 1-15 for the manufacture of a medicament for depleting B cells in a subject. The monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 15 , for depleting B cells in a subject. Use of the monoclonal antibody or antigen-binding portion thereof according to any of claims 1-15 for the manufacture of a medicament for treating an autoimmune disease in a subject. The use according to claim 32 , wherein the autoimmune disease is rheumatoid arthritis. The monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 15 , for treating an autoimmune disease in a subject.

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